Methods and devices for electronic sensing

ABSTRACT

The presence of a detectable entity within a detection volume of a microfabricated elastomeric structure is sensed through a change in the electrical or magnetic environment of the detection volume. In embodiments utilizing electronic detection, an electric field is applied to the detection volume and a change in impedance, current, or combined impedance and current due to the presence of the detectable entity is measured. In embodiments utilizing magnetic detection, the magnetic properties of a magnetized detected entity alter the magnetic field of the detection volume. This changed magnetic field induces a current which can reveal the detectable entity. The change in resistance of a magnetoresistive element may also reveal the passage of a magnetized detectable entity.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/867,555, filed Apr. 22, 2013, and U.S. patent application Ser. No.11/546,939, filed Oct. 11, 2006, now U.S. Pat. No. 8,440,093. The '555application is a continuation of U.S. patent application Ser. No.11/546,939, filed Oct. 11, 2006, now U.S. Pat. No. 8,440,093. The '939application is a continuation of U.S. patent application Ser. No.10/273,406, filed Oct. 16, 2002, which claims the priority benefit ofU.S. Prov. Pat. Appl. No. 60/348,448, filed Oct. 26, 2001. The foregoingapplications are hereby incorporated herein by reference in theirentireties for all purposes.

BACKGROUND OF THE INVENTION

U.S. patent application Ser. No. 09/605,520 (“the '520 application”)describes in detail the use of elastomeric materials to fabricatemicrofluidic structures. The '520 application is hereby incorporated byreference in its entirety for all purposes.

The microfabricated elastomeric structures disclosed in the '520application may be employed for a wide variety of purposes. Oneapplication for which these structures are particularly suited issorting. In a sorting structure, a sample containing a sortable entityis flowed down a flow channel to a detection region, such that only onesortable entity may be located within the detection region at a time.The detection region is then interrogated to identify the sortableentity. The sortable entity is then flowed to a junction, and then downone or another branch at the junction based upon the identificationprocess.

As described in the '520 application, the width of the flow channels maybe defined utilizing photolithographic techniques conventionallyemployed in semiconductor fabrication processes. Accordingly, thedimensions of the flow channels may extremely small (<1 μm), allowingfor sorting of entities on the cellular or molecular scale.

One of the most important steps of a sorting process is the accuratedetection and identification of an entity prior to its sorting. Thisdetection/identification task is made more difficult when the entity isextremely small.

Accordingly, there is a need in the art for methods and structures fordetecting and identifying the contents of the extremely narrow flowchannels of microfluidic devices.

BRIEF SUMMARY OF THE INVENTION

The presence of a detectable entity within a detection region of amicrofabricated elastomeric structure is detected through a change inthe electrical or magnetic environment of the detection region. Inembodiments utilizing electronic detection, an electric field is appliedto the detection region and a change in impedance or current due to thepresence of the detectable entity is measured. In embodiments utilizingmagnetic detection, the magnetic properties of a detected entity alterthe magnetic field of the detection volume and can be sensed by inducedcurrents, changed electric fields, or changes in the behavior ofmagnetoresistive elements.

An embodiment of a method of detecting an entity in a microfabricatedelastomeric structure comprises providing a microfabricated elastomericstructure including a flow channel and a deflectable elastomericmembrane. Defined within the flow channel is a detection volumereceiving one detectable entity or an ensemble of detected entities at atime. An electric field is applied to the detection volume, and a changeis measured in one of a voltage across and a current through thedetection volume as the detectable entity traverses the detectionvolume.

An embodiment of a method of detecting an entity in a microfabricatedelastomeric structure comprises providing the microfabricatedelastomeric structure including a flow channel and a deflectableelastomeric membrane. A detection volume is defined within the flowchannel, the detection volume receiving one magnetized detectable entityor an ensemble of magnetized detected entities at a time. A conductivecoil is provided proximate to the detection volume, and a currentinduced in the coil structure by passage of the magnetized detectableentity through the detection volume is measured.

An embodiment of a microfabricated elastomeric structure in accordancewith the present invention comprises a flow channel formed in anelastomeric material. A control recess overlies and is separated fromthe flow channel by an elastomeric membrane deflectable into the flowchannel. A detection volume is defined within the flow channel toreceive one detectable entity or ensemble of detected entities at atime. A first electrode and a second electrode formed in the elastomermaterial are in communication with a power supply and configured toapply an electric field to the detection volume.

These and other embodiments of the present invention, as well as itsadvantages and features, are described in more detail in conjunctionwith the text below and attached figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a first elastomeric layer formed on top ofa micromachined mold.

FIG. 2 is an illustration of a second elastomeric layer formed on top ofa micromachined mold.

FIG. 3 is an illustration of the elastomeric layer of FIG. 2 removedfrom the micromachined mold and positioned over the top of theelastomeric layer of FIG. 1

FIG. 4 is an illustration corresponding to FIG. 3, but showing thesecond elastomeric layer positioned on top of the first elastomericlayer.

FIG. 5 is an illustration corresponding to FIG. 4, but showing the firstand second elastomeric layers bonded together.

FIG. 6 is an illustration corresponding to FIG. 5, but showing the firstmicromachined mold removed and a planar substrate positioned in itsplace.

FIG. 7A is an illustration corresponding to FIG. 6, but showing theelastomeric structure sealed onto the planar substrate.

FIG. 7B is a front sectional view corresponding to FIG. 7A, showing anopen flow channel.

FIGS. 7C to 7G are illustrations showing steps of a method for formingan elastomeric structure having a membrane formed from a separateelastomeric layer. FIG. 7H shows first flow channel 30 closed bypressurization of the second flow channel 32.

FIGS. 8A and 8B illustrates valve opening vs. applied pressure forvarious flow channels.

FIG. 9 illustrates time response of a 100 μm×100 μm×10 μm RTVmicrovalve.

FIG. 10 is a cross sectional view through flow channels 30 and 32.

FIG. 11 is an alternative embodiment in which flow channel 30A has acurved upper wall 25A.

FIG. 12A is a top schematic view of an on/off valve.

FIG. 12B is a sectional elevation view along line 23B-23B in FIG. 12A

FIG. 13A is a top schematic view of a peristaltic pumping system.

FIG. 13B is a sectional elevation view along line 24B-24B in FIG. 13A

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for an embodiment of the peristaltic pumping system of FIG.13.

FIG. 15A is a top schematic view of one control line actuating multipleflow lines simultaneously.

FIG. 15B is a sectional elevation view along line 26B-26B in FIG. 15A

FIG. 16 is a schematic illustration of a multiplexed system adapted topermit flow through various channels.

FIG. 17A is a plan view of a flow layer of an addressable reactionchamber structure.

FIG. 17B is a bottom plan view of a control channel layer of anaddressable reaction chamber structure.

FIG. 17C is an exploded perspective view of the addressable reactionchamber structure formed by bonding the control channel layer of FIG.17B to the top of the flow layer of FIG. 17A.

FIG. 17D is a sectional elevation view corresponding to FIG. 17C, takenalong line 28D-28D in FIG. 17C.

FIG. 18 is a schematic of a system adapted to selectively direct fluidflow into any of an array of reaction wells.

FIG. 19 is a schematic of a system adapted for selectable lateral flowbetween parallel flow channels.

FIG. 20A is a bottom plan view of first layer (i.e.: the flow channellayer) of elastomer of a switchable flow array.

FIG. 20B is a bottom plan view of a control channel layer of aswitchable flow array.

FIG. 20C shows the alignment of the first layer of elastomer of FIG. 20Awith one set of control channels in the second layer of elastomer ofFIG. 20B.

FIG. 20D also shows the alignment of the first layer of elastomer ofFIG. 20A with the other set of control channels in the second layer ofelastomer of FIG. 20B.

FIGS. 21A-21J show views of one embodiment of a normally-closed valvestructure in accordance with the present invention.

FIGS. 22A and 22B show plan views illustrating operation of oneembodiment of a side-actuated valve structure in accordance with thepresent invention.

FIG. 23 shows a cross-sectional view of one embodiment of a compositestructure in accordance with the present invention.

FIG. 24 shows a cross-sectional view of another embodiment of acomposite structure in accordance with the present invention.

FIG. 25 shows a cross-sectional view of another embodiment of acomposite structure in accordance with the present invention.

FIGS. 26A-26D show plan views illustrating operation of one embodimentof a cell pen structure in accordance with the present invention.

FIGS. 27A-27B show plan and cross-sectional views illustrating operationof one embodiment of a cell cage structure in accordance with thepresent invention.

FIGS. 28A-28B show plan views of operation of a wiring structureutilizing cross-channel injection in accordance with the embodiment ofthe present invention.

FIGS. 29A-29D illustrate cross-sectional views of metering by volumeexclusion in accordance with an embodiment of the present invention.

FIG. 30 illustrates a plan view of one embodiment of a sorting device inaccordance with the present invention.

FIG. 31A shows a simplified plan view of the detection region of anembodiment of a “transverse” electronic-based detection apparatus inaccordance with the present invention.

FIGS. 31B-C show perspective views of alternative embodiments ofdetection regions of a “transverse” electronic-based detectionapparatuses.

FIG. 32 shows a simplified plan view of the detection region of anembodiment of a “longitudinal” electronic-based detection apparatus inaccordance with the present invention.

FIG. 33A shows a simplified plan view of an alternative embodiment of a“transverse” electronic-based detection apparatus in accordance with thepresent invention.

FIG. 33B shows a schematic diagram of the circuit formed by theapparatus of FIG. 33A.

FIG. 34 shows a simplified plan view of an alternative embodiment of a“longitudinal” electronic-based detection apparatus in accordance withthe present invention.

FIG. 35 shows a simplified plan view of another alternative embodimentof a “longitudinal” electronic-based detection apparatus in accordancewith the present invention.

FIG. 36 shows a simplified plan view of an embodiment of amagnetic-based detection apparatus in accordance with the presentinvention.

FIG. 37 shows a simplified perspective view of an alternative embodimentof a magnetic-based detection apparatus in accordance with the presentinvention.

FIG. 38 shows a simplified plan view of another alternative embodimentof a magnetic-based detection apparatus in accordance with the presentinvention.

FIG. 39 shows a simplified plan view of still another alternativeembodiment of a magnetic-based detection apparatus in accordance withthe present invention.

FIG. 40 shows a simplified plan view of a detection region in accordancewith an alternative embodiment of the present invention

DETAILED DESCRIPTION OF THE INVENTION

I. Microfabrication Overview

The following discussion relates to formation of microfabricated fluidicdevices utilizing elastomer materials, as described generally in U.S.patent application Ser. No. 09/826,585 filed Apr. 6, 2001, Ser. No.09/724,784 filed Nov. 28, 2000, and Ser. No. 09/605,520, filed Jun. 27,2000. These patent applications are hereby incorporated by reference.

1. Methods of Fabricating

Exemplary methods of fabricating the present invention are providedherein. It is to be understood that the present invention is not limitedto fabrication by one or the other of these methods. Rather, othersuitable methods of fabricating the present microstructures, includingmodifying the present methods, are also contemplated.

FIGS. 1 to 7B illustrate sequential steps of a first preferred method offabricating the present microstructure, (which may be used as a pump orvalve). FIGS. 8 to 18 illustrate sequential steps of a second preferredmethod of fabricating the present microstructure, (which also may beused as a pump or valve).

As will be explained, the preferred method of FIGS. 1 to 7B involvesusing pre-cured elastomer layers which are assembled and bonded. In analternative method, each layer of elastomer may be cured “in place”. Inthe following description “channel” refers to a recess in theelastomeric structure which can contain a flow of fluid or gas.

Referring to FIG. 1, a first micro-machined mold 10 is provided.Micro-machined mold 10 may be fabricated by a number of conventionalsilicon processing methods, including but not limited tophotolithography, ion-milling, and electron beam lithography.

As can be seen, micro-machined mold 10 has a raised line or protrusion11 extending therealong. A first elastomeric layer 20 is cast on top ofmold 10 such that a first recess 21 will be formed in the bottom surfaceof elastomeric layer 20, (recess 21 corresponding in dimension toprotrusion 11), as shown.

As can be seen in FIG. 2, a second micro-machined mold 12 having araised protrusion 13 extending therealong is also provided. A secondelastomeric layer 22 is cast on top of mold 12, as shown, such that arecess 23 will be formed in its bottom surface corresponding to thedimensions of protrusion 13.

As can be seen in the sequential steps illustrated in FIGS. 3 and 4,second elastomeric layer 22 is then removed from mold 12 and placed ontop of first elastomeric layer 20. As can be seen, recess 23 extendingalong the bottom surface of second elastomeric layer 22 will form a flowchannel 32.

Referring to FIG. 5, the separate first and second elastomeric layers 20and 22 (FIG. 4) are then bonded together to form an integrated (i.e.:monolithic) elastomeric structure 24.

As can been seen in the sequential step of FIGS. 6 and 7A, elastomericstructure 24 is then removed from mold 10 and positioned on top of aplanar substrate 14. As can be seen in FIGS. 7A and 7B, when elastomericstructure 24 has been sealed at its bottom surface to planar substrate14, recess 21 will form a flow channel 30.

The present elastomeric structures form a reversible hermetic seal withnearly any smooth planar substrate. An advantage to forming a seal thisway is that the elastomeric structures may be peeled up, washed, andre-used. In preferred aspects, planar substrate 14 is glass. A furtheradvantage of using glass is that glass is transparent, allowing opticalinterrogation of elastomer channels and reservoirs. Alternatively, theelastomeric structure may be bonded onto a flat elastomer layer by thesame method as described above, forming a permanent and high-strengthbond. This may prove advantageous when higher back pressures are used.

As can be seen in FIGS. 7A and 7B, flow channels 30 and 32 arepreferably disposed at an angle to one another with a small membrane 25of substrate 24 separating the top of flow channel 30 from the bottom offlow channel 32.

In preferred aspects, planar substrate 14 is glass. An advantage ofusing glass is that the present elastomeric structures may be peeled up,washed and reused. A further advantage of using glass is that opticalsensing may be employed. Alternatively, planar substrate 14 may be anelastomer itself, which may prove advantageous when higher backpressures are used.

The method of fabrication just described may be varied to form astructure having a membrane composed of an elastomeric materialdifferent than that forming the walls of the channels of the device.This variant fabrication method is illustrated in FIGS. 7C-7G.

Referring to FIG. 7C, a first micro-machined mold 10 is provided.Micro-machined mold 10 has a raised line or protrusion 11 extendingtherealong. In FIG. 7D, first elastomeric layer 20 is cast on top offirst micro-machined mold 10 such that the top of the first elastomericlayer 20 is flush with the top of raised line or protrusion 11. This maybe accomplished by carefully controlling the volume of elastomericmaterial spun onto mold 10 relative to the known height of raised line11. Alternatively, the desired shape could be formed by injectionmolding.

In FIG. 7E, second micro-machined mold 12 having a raised protrusion 13extending therealong is also provided. Second elastomeric layer 22 iscast on top of second mold 12 as shown, such that recess 23 is formed inits bottom surface corresponding to the dimensions of protrusion 13.

In FIG. 7F, second elastomeric layer 22 is removed from mold 12 andplaced on top of third elastomeric layer 222. Second elastomeric layer22 is bonded to third elastomeric layer 20 to form integral elastomericblock 224 using techniques described in detail below. At this point inthe process, recess 23 formerly occupied by raised line 13 will formflow channel 23.

In FIG. 7G, elastomeric block 224 is placed on top of firstmicro-machined mold 10 and first elastomeric layer 20. Elastomeric blockand first elastomeric layer 20 are then bonded together to form anintegrated (i.e.: monolithic) elastomeric structure 24 having a membranecomposed of a separate elastomeric layer 222.

When elastomeric structure 24 has been sealed at its bottom surface to aplanar substrate in the manner described above in connection with FIG.7A, the recess formerly occupied by raised line 11 will form flowchannel 30.

The variant fabrication method illustrated above in conjunction withFIGS. 7C-7G offers the advantage of permitting the membrane portion tobe composed of a separate material than the elastomeric material of theremainder of the structure. This is important because the thickness andelastic properties of the membrane play a key role in operation of thedevice. Moreover, this method allows the separate elastomer layer toreadily be subjected to conditioning prior to incorporation into theelastomer structure. As discussed in detail below, examples ofpotentially desirable condition include the introduction of magnetic orelectrically conducting species to permit actuation of the membrane,and/or the introduction of dopant into the membrane in order to alterits elasticity.

While the above method is illustrated in connection with forming variousshaped elastomeric layers formed by replication molding on top of amicromachined mold, the present invention is not limited to thistechnique. Other techniques could be employed to form the individuallayers of shaped elastomeric material that are to be bonded together.For example, a shaped layer of elastomeric material could be formed bylaser cutting or injection molding, or by methods utilizing chemicaletching and/or sacrificial materials as discussed below in conjunctionwith the second exemplary method.

An alternative method fabricates a patterned elastomer structureutilizing development of photoresist encapsulated within elastomermaterial. However, the methods in accordance with the present inventionare not limited to utilizing photoresist. Other materials such as metalscould also serve as sacrificial materials to be removed selective to thesurrounding elastomer material, and the method would remain within thescope of the present invention. For example, gold metal may be etchedselective to RTV 615 elastomer utilizing the appropriate chemicalmixture.

2. Layer and Channel Dimensions

Microfabricated refers to the size of features of an elastomericstructure fabricated in accordance with an embodiment of the presentinvention. In general, variation in at least one dimension ofmicrofabricated structures is controlled to the micron level, with atleast one dimension being microscopic (i.e. below 1000 μm).Microfabrication typically involves semiconductor or MEMS fabricationtechniques such as photolithography and spincoating that are designedfor to produce feature dimensions on the microscopic level, with atleast some of the dimension of the microfabricated structure requiring amicroscope to reasonably resolve/image the structure.

In preferred aspects, flow channels 30, 32, 60 and 62 preferably havewidth-to-depth ratios of about 10:1. A non-exclusive list of otherranges of width-to-depth ratios in accordance with embodiments of thepresent invention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplaryaspect, flow channels 30, 32, 60 and 62 have widths of about 1 to 1000microns. A non-exclusive list of other ranges of widths of flow channelsin accordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to500 microns, more preferably 1 to 250 microns, and most preferably 10 to200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm,200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.

Flow channels 30, 32, 60, and 62 have depths of about 1 to 100 microns.A non-exclusive list of other ranges of depths of flow channels inaccordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250microns, and more preferably 1 to 100 microns, more preferably 2 to 20microns, and most preferably 5 to 10 microns. Exemplary channel depthsinclude including 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1μm, 2 μm, 3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm,and 250 μm.

The flow channels are not limited to these specific dimension ranges andexamples given above, and may vary in width in order to affect themagnitude of force required to deflect the membrane as discussed atlength below in conjunction with FIG. 27. For example, extremely narrowflow channels having a width on the order of 0.01 μm may be useful inoptical and other applications, as discussed in detail below.Elastomeric structures which include portions having channels of evengreater width than described above are also contemplated by the presentinvention, and examples of applications of utilizing such wider flowchannels include fluid reservoir and mixing channel structures.

The Elastomeric layers may be cast thick for mechanical stability. In anexemplary embodiment, elastomeric layer 22 of FIG. 1 is 50 microns toseveral centimeters thick, and more preferably approximately 4 mm thick.A non-exclusive list of ranges of thickness of the elastomer layer inaccordance with other embodiments of the present invention is betweenabout 0.1 micron to 10 cm, 1 micron to 5 cm, 10 microns to 2 cm, 100microns to 10 mm.

Accordingly, membrane 25 of FIG. 7B separating flow channels 30 and 32has a typical thickness of between about 0.01 and 1000 microns, morepreferably 0.05 to 500 microns, more preferably 0.2 to 250, morepreferably 1 to 100 microns, more preferably 2 to 50 microns, and mostpreferably 5 to 40 microns. As such, the thickness of elastomeric layer22 is about 100 times the thickness of elastomeric layer 20. Exemplarymembrane thicknesses include 0.01 μm, 0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm,0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm,15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 750 μm, and 1000 μm.

3. Soft Lithographic Bonding

Preferably, elastomeric layers are bonded together chemically, usingchemistry that is intrinsic to the polymers comprising the patternedelastomer layers. Most preferably, the bonding comprises two component“addition cure” bonding.

In a preferred aspect, the various layers of elastomer are boundtogether in a heterogenous bonding in which the layers have a differentchemistry. Alternatively, a homogenous bonding may be used in which alllayers would be of the same chemistry. Thirdly, the respective elastomerlayers may optionally be glued together by an adhesive instead. In afourth aspect, the elastomeric layers may be thermoset elastomers bondedtogether by heating.

In one aspect of homogeneous bonding, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In one embodiment, bonding between polymerchains of like elastomer layers may result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

Alternatively in a heterogeneous aspect, the elastomeric layers arecomposed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. In one exemplary heterogenous aspect, the bonding process used tobind respective elastomeric layers together may comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer may bemade with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B(i.e. excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

In an exemplary aspect of the present invention, elastomeric structuresare formed utilizing Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCBChemical.

In one embodiment in accordance with the present invention, two-layerelastomeric structures were fabricated from pure acrylated Urethane Ebe270. A thin bottom layer was spin coated at 8000 rpm for 15 seconds at170° C. The top and bottom layers were initially cured under ultravioletlight for 10 minutes under nitrogen utilizing a Model ELC 500 devicemanufactured by Electrolyte corporation. The assembled layers were thencured for an additional 30 minutes. Reaction was catalyzed by a 0.5%vol./vol. mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals.The resulting elastomeric material exhibited moderate elasticity andadhesion to glass.

In another embodiment in accordance with the present invention,two-layer elastomeric structures were fabricated from a combination of25% Ebe 270/50% Irr 245/25% isopropyl alcohol for a thin bottom layer,and pure acrylated Urethane Ebe 270 as a top layer. The thin bottomlayer was initially cured for 5 min, and the top layer initially curedfor 10 minutes, under ultraviolet light under nitrogen utilizing a ModelELC 500 device manufactured by Electrolyte corporation. The assembledlayers were then cured for an additional 30 minutes. Reaction wascatalyzed by a 0.5% vol./vol. mixture of Irgacure 500 manufactured byCiba-Geigy Chemicals. The resulting elastomeric material exhibitedmoderate elasticity and adhered to glass.

Alternatively, other bonding methods may be used, including activatingthe elastomer surface, for example by plasma exposure, so that theelastomer layers/substrate will bond when placed in contact. Forexample, one possible approach to bonding together elastomer layerscomposed of the same material is set forth by Duffy et al, “RapidPrototyping of Microfluidic Systems in Poly (dimethylsiloxane)”,Analytical Chemistry (1998), 70, 4974-4984, incorporated herein byreference. This paper discusses that exposing polydimethylsiloxane(PDMS) layers to oxygen plasma causes oxidation of the surface, withirreversible bonding occurring when the two oxidized layers are placedinto contact.

Yet another approach to bonding together successive layers of elastomeris to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured elastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

Where encapsulation of sacrificial layers is employed to fabricate theelastomer structure, bonding of successive elastomeric layers may beaccomplished by pouring uncured elastomer over a previously curedelastomeric layer and any sacrificial material patterned thereupon.Bonding between elastomer layers occurs due to interpenetration andreaction of the polymer chains of an uncured elastomer layer with thepolymer chains of a cured elastomer layer. Subsequent curing of theelastomeric layer will create a bond between the elastomeric layers andcreate a monolithic elastomeric structure.

Referring to the first method of FIGS. 1 to 7B, first elastomeric layer20 may be created by spin-coating an RTV mixture on microfabricated mold12 at 2000 rpm's for 30 seconds yielding a thickness of approximately 40microns. Second elastomeric layer 22 may be created by spin-coating anRTV mixture on microfabricated mold 11. Both layers 20 and 22 may beseparately baked or cured at about 80° C. for 1.5 hours. The secondelastomeric layer 22 may be bonded onto first elastomeric layer 20 atabout 80° C. for about 1.5 hours.

Micromachined molds 10 and 12 may be patterned photoresist on siliconwafers. In an exemplary aspect, a Shipley SJR 5740 photoresist was spunat 2000 rpm patterned with a high resolution transparency film as a maskand then developed yielding an inverse channel of approximately 10microns in height. When baked at approximately 200° C. for about 30minutes, the photoresist reflows and the inverse channels becomerounded. In preferred aspects, the molds may be treated withtrimethylchlorosilane (TMCS) vapor for about a minute before each use inorder to prevent adhesion of silicone rubber.

4. Suitable Elastomeric Materials

Allcock et al, Contemporary Polymer Chemistry, 2^(nd) Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.Elastomeric materials having a Young's modulus of between about 1 Pa-1TPa, more preferably between about 10 Pa-100 GPa, more preferablybetween about 20 Pa-1 GPa, more preferably between about 50 Pa-10 MPa,and more preferably between about 100 Pa-1 MPa are useful in accordancewith the present invention, although elastomeric materials having aYoung's modulus outside of these ranges could also be utilized dependingupon the needs of a particular application.

The systems of the present invention may be fabricated from a widevariety of elastomers. In an exemplary aspect, the elastomeric layersmay preferably be fabricated from silicone rubber. However, othersuitable elastomers may also be used.

In an exemplary aspect of the present invention, the present systems arefabricated from an elastomeric polymer such as GE RTV 615 (formulation),a vinyl-silane crosslinked (type) silicone elastomer (family). However,the present systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. An important requirement for the preferred method offabrication of the present microvalves is the ability to bond multiplelayers of elastomers together. In the case of multilayer softlithography, layers of elastomer are cured separately and then bondedtogether. This scheme requires that cured layers possess sufficientreactivity to bond together. Either the layers may be of the same type,and are capable of bonding to themselves, or they may be of twodifferent types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microvalves and pumps. Variations in thematerials used will most likely be driven by the need for particularmaterial properties, i.e. solvent resistance, stiffness, gaspermeability, or temperature stability.

There are many, many types of elastomeric polymers. A brief descriptionof the most common classes of elastomers is presented here, with theintent of showing that even with relatively “standard” polymers, manypossibilities for bonding exist. Common elastomeric polymers includepolyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones.

Polyisoprene, Polybutadiene, Polychloroprene:

-   -   Polyisoprene, polybutadiene, and polychloroprene are all        polymerized from diene monomers, and therefore have one double        bond per monomer when polymerized. This double bond allows the        polymers to be converted to elastomers by vulcanization        (essentially, sulfur is used to form crosslinks between the        double bonds by heating). This would easily allow homogeneous        multilayer soft lithography by incomplete vulcanization of the        layers to be bonded; photoresist encapsulation would be possible        by a similar mechanism.

Polyisobutylene:

-   -   Pure polyisobutylene has no double bonds, but is crosslinked to        use as an elastomer by including a small amount (˜1%) of        isoprene in the polymerization. The isoprene monomers give        pendant double bonds on the polyisobutylene backbone, which may        then be vulcanized as above.

Poly(Styrene-Butadiene-Styrene):

-   -   Poly(styrene-butadiene-styrene) is produced by living anionic        polymerization (that is, there is no natural chain-terminating        step in the reaction), so “live” polymer ends can exist in the        cured polymer. This makes it a natural candidate for the present        photoresist encapsulation system (where there will be plenty of        unreacted monomer in the liquid layer poured on top of the cured        layer). Incomplete curing would allow homogeneous multilayer        soft lithography (A to A bonding). The chemistry also        facilitates making one layer with extra butadiene (“A”) and        coupling agent and the other layer (“B”) with a butadiene        deficit (for heterogeneous multilayer soft lithography). SBS is        a “thermoset elastomer”, meaning that above a certain        temperature it melts and becomes plastic (as opposed to        elastic); reducing the temperature yields the elastomer again.        Thus, layers can be bonded together by heating.

Polyurethane's:

-   -   Polyurethane's are produced from di-isocyanates (A-A) and        di-alcohols or di-amines (B-B); since there are a large variety        of di-isocyanates and di-alcohols/amines, the number of        different types of polyurethanes is huge. The A vs. B nature of        the polymers, however, would make them useful for heterogeneous        multilayer soft lithography just as RTV 615 is: by using excess        A-A in one layer and excess B-B in the other layer.

Silicones:

-   -   Silicone polymers probably have the greatest structural variety,        and almost certainly have the greatest number of commercially        available formulations. The vinyl-to-(Si—H) crosslinking of RTV        615 (which allows both heterogeneous multilayer soft lithography        and photoresist encapsulation) has already been discussed, but        this is only one of several crosslinking methods used in        silicone polymer chemistry.

5. Operation of Device

FIGS. 7B and 7H together show the closing of a first flow channel bypressurizing a second flow channel, with FIG. 7B (a front sectional viewcutting through flow channel 32 in corresponding FIG. 7A), showing anopen first flow channel 30; with FIG. 7H showing first flow channel 30closed by pressurization of the second flow channel 32.

Referring to FIG. 7B, first flow channel 30 and second flow channel 32are shown. Membrane 25 separates the flow channels, forming the top offirst flow channel 30 and the bottom of second flow channel 32. As canbe seen, flow channel 30 is “open”.

As can be seen in FIG. 7H, pressurization of flow channel 32 (either bygas or liquid introduced therein) causes membrane 25 to deflectdownward, thereby pinching off flow F passing through flow channel 30.Accordingly, by varying the pressure in channel 32, a linearly actuablevalving system is provided such that flow channel 30 can be opened orclosed by moving membrane 25 as desired. (For illustration purposesonly, channel 30 in FIG. 7G is shown in a “mostly closed” position,rather than a “fully closed” position).

Since such valves are actuated by moving the roof of the channelsthemselves (i.e.: moving membrane 25) valves and pumps produced by thistechnique have a truly zero dead volume, and switching valves made bythis technique have a dead volume approximately equal to the activevolume of the valve, for example about 100×100×10 μm=100 pL. Such deadvolumes and areas consumed by the moving membrane are approximately twoorders of magnitude smaller than known conventional microvalves. Smallerand larger valves and switching valves are contemplated in the presentinvention, and a non-exclusive list of ranges of dead volume includes 1aL to 1 uL, 100 aL to 100 nL, 1 fL to 10 nL, 100 fL to 1 nL, and 1 pL to100 pL

The extremely small volumes capable of being delivered by pumps andvalves in accordance with the present invention represent a substantialadvantage. Specifically, the smallest known volumes of fluid capable ofbeing manually metered is around 0.1 μl. The smallest known volumescapable of being metered by automated systems is about ten-times larger(1 μl). Utilizing pumps and valves in accordance with the presentinvention, volumes of liquid of 10 nl or smaller can routinely bemetered and dispensed. The accurate metering of extremely small volumesof fluid enabled by the present invention would be extremely valuable ina large number of biological applications, including diagnostic testsand assays.

Equation 1 represents a highly simplified mathematical model ofdeflection of a rectangular, linear, elastic, isotropic plate of uniformthickness by an applied pressure:w=(BPb ⁴)/(Eh ³),where:  (1)

-   -   w=deflection of plate;    -   B=shape coefficient (dependent upon length vs. width and support        of edges of plate);    -   P=applied pressure;    -   b=plate width    -   E=Young's modulus; and    -   h=plate thickness.        Thus even in this extremely simplified expression, deflection of        an elastomeric membrane in response to a pressure will be a        function of: the length, width, and thickness of the membrane,        the flexibility of the membrane (Young's modulus), and the        applied actuation force. Because each of these parameters will        vary widely depending upon the actual dimensions and physical        composition of a particular elastomeric device in accordance        with the present invention, a wide range of membrane thicknesses        and elasticities, channel widths, and actuation forces are        contemplated by the present invention.

It should be understood that the formula just presented is only anapproximation, since in general the membrane does not have uniformthickness, the membrane thickness is not necessarily small compared tothe length and width, and the deflection is not necessarily smallcompared to length, width, or thickness of the membrane. Nevertheless,the equation serves as a useful guide for adjusting variable parametersto achieve a desired response of deflection versus applied force.

FIGS. 8A and 8B illustrate valve opening vs. applied pressure for a 100nm wide first flow channel 30 and a 50 nm wide second flow channel 32.The membrane of this device was formed by a layer of General ElectricSilicones RTV 615 having a thickness of approximately 30 μm and aYoung's modulus of approximately 750 kPa. FIGS. 21 a and 21 b show theextent of opening of the valve to be substantially linear over most ofthe range of applied pressures.

Air pressure was applied to actuate the membrane of the device through a10 cm long piece of plastic tubing having an outer diameter of 0.025″connected to a 25 mm piece of stainless steel hypodermic tubing with anouter diameter of 0.025″ and an inner diameter of 0.013″. This tubingwas placed into contact with the control channel by insertion into theelastomeric block in a direction normal to the control channel. Airpressure was applied to the hypodermic tubing from an external LHDAminiature solenoid valve manufactured by Lee Co.

While control of the flow of material through the device has so far beendescribed utilizing applied gas pressure, other fluids could be used.

For example, air is compressible, and thus experiences some finite delaybetween the time of application of pressure by the external solenoidvalve and the time that this pressure is experienced by the membrane. Inan alternative embodiment of the present invention, pressure could beapplied from an external source to a noncompressible fluid such as wateror hydraulic oils, resulting in a near-instantaneous transfer of appliedpressure to the membrane. However, if the displaced volume of the valveis large or the control channel is narrow, higher viscosity of a controlfluid may contribute to delay in actuation. The optimal medium fortransferring pressure will therefore depend upon the particularapplication and device configuration, and both gaseous and liquid mediaare contemplated by the invention.

While external applied pressure as described above has been applied by apump/tank system through a pressure regulator and external miniaturevalve, other methods of applying external pressure are also contemplatedin the present invention, including gas tanks, compressors, pistonsystems, and columns of liquid. Also contemplated is the use ofnaturally occurring pressure sources such as may be found inside livingorganisms, such as blood pressure, gastric pressure, the pressurepresent in the cerebro-spinal fluid, pressure present in theintra-ocular space, and the pressure exerted by muscles during normalflexure. Other methods of regulating external pressure are alsocontemplated, such as miniature valves, pumps, macroscopic peristalticpumps, pinch valves, and other types of fluid regulating equipment suchas is known in the art.

As can be seen, the response of valves in accordance with embodiments ofthe present invention have been experimentally shown to be almostperfectly linear over a large portion of its range of travel, withminimal hysteresis. Accordingly, the present valves are ideally suitedfor microfluidic metering and fluid control. The linearity of the valveresponse demonstrates that the individual valves are well modeled asHooke's Law springs. Furthermore, high pressures in the flow channel(i.e.: back pressure) can be countered simply by increasing theactuation pressure. Experimentally, the present inventors have achievedvalve closure at back pressures of 70 kPa, but higher pressures are alsocontemplated. The following is a nonexclusive list of pressure rangesencompassed by the present invention: 10 Pa-25 MPa; 100 Pa-10 Mpa, 1kPa-1 MPa, 1 kPa-300 kPa, 5 kPa-200 kPa, and 15 kPa-100 kPa.

While valves and pumps do not require linear actuation to open andclose, linear response does allow valves to more easily be used asmetering devices. In one embodiment of the invention, the opening of thevalve is used to control flow rate by being partially actuated to aknown degree of closure. Linear valve actuation makes it easier todetermine the amount of actuation force required to close the valve to adesired degree of closure. Another benefit of linear actuation is thatthe force required for valve actuation may be easily determined from thepressure in the flow channel. If actuation is linear, increased pressurein the flow channel may be countered by adding the same pressure (forceper unit area) to the actuated portion of the valve.

Linearity of a valve depends on the structure, composition, and methodof actuation of the valve structure. Furthermore, whether linearity is adesirable characteristic in a valve depends on the application.Therefore, both linearly and non-linearly actuable valves arecontemplated in the present invention, and the pressure ranges overwhich a valve is linearly actuable will vary with the specificembodiment.

FIG. 9 illustrates time response (i.e.: closure of valve as a functionof time in response to a change in applied pressure) of a 100 μm×100μm×10 μm RTV microvalve with 10-cm-long air tubing connected from thechip to a pneumatic valve as described above.

Two periods of digital control signal, actual air pressure at the end ofthe tubing and valve opening are shown in FIG. 9. The pressure appliedon the control line is 100 kPa, which is substantially higher than the˜40 kPa required to close the valve. Thus, when closing, the valve ispushed closed with a pressure 60 kPa greater than required. Whenopening, however, the valve is driven back to its rest position only byits own spring force (≦40 kPa). Thus, τ_(close) is expected to besmaller than τ_(open). There is also a lag between the control signaland control pressure response, due to the limitations of the miniaturevalve used to control the pressure. Calling such lags t and the 1/e timeconstants τ, the values are: t_(open)=3.63 ms, τ_(open)=1.88 ms,t_(close)=2.15 ms, τ_(close)=0.51 ms. If 3τ each are allowed for openingand closing, the valve runs comfortably at 75 Hz when filled withaqueous solution.

If one used another actuation method which did not suffer from openingand closing lag, this valve would run at ˜375 Hz. Note also that thespring constant can be adjusted by changing the membrane thickness; thisallows optimization for either fast opening or fast closing. The springconstant could also be adjusted by changing the elasticity (Young'smodulus) of the membrane, as is possible by introducing dopant into themembrane or by utilizing a different elastomeric material to serve asthe membrane (described above in conjunction with FIGS. 7C-7H.)

When experimentally measuring the valve properties as illustrated inFIG. 9 the valve opening was measured by fluorescence. In theseexperiments, the flow channel was filled with a solution of fluoresceinisothiocyanate (FITC) in buffer (pH≧8) and the fluorescence of a squarearea occupying the center ˜⅓rd of the channel is monitored on anepi-fluorescence microscope with a photomultiplier tube with a 10 kHzbandwidth. The pressure was monitored with a Wheatstone-bridge pressuresensor (SenSym SCC15GD2) pressurized simultaneously with the controlline through nearly identical pneumatic connections.

6. Flow Channel Cross Sections

The flow channels of the present invention may optionally be designedwith different cross sectional sizes and shapes, offering differentadvantages, depending upon their desired application. For example, thecross sectional shape of the lower flow channel may have a curved uppersurface, either along its entire length or in the region disposed underan upper cross channel). Such a curved upper surface facilitates valvesealing, as follows.

Referring to FIG. 10, a cross sectional view (similar to that of FIG.7B) through flow channels 30 and 32 is shown. As can be seen, flowchannel 30 is rectangular in cross sectional shape. In an alternatepreferred aspect of the invention, as shown in FIG. 20, thecross-section of a flow channel 30 instead has an upper curved surface.

Referring first to FIG. 10, when flow channel 32 is pressurized, themembrane portion 25 of elastomeric block 24 separating flow channels 30and 32 will move downwardly to the successive positions shown by thedotted lines 25A, 25B, 25C, 25D, and 25E. As can be seen, incompletesealing may possibly result at the edges of flow channel 30 adjacentplanar substrate 14.

In the alternate preferred embodiment of FIG. 11, flow channel 30 a hasa curved upper wall 25A. When flow channel 32 is pressurized, membraneportion 25 will move downwardly to the successive positions shown bydotted lines 25A2, 25A3, 25A4 and 25A5, with edge portions of themembrane moving first into the flow channel, followed by top membraneportions. An advantage of having such a curved upper surface at membrane25A is that a more complete seal will be provided when flow channel 32is pressurized. Specifically, the upper wall of the flow channel 30 willprovide a continuous contacting edge against planar substrate 14,thereby avoiding the “island” of contact seen between wall 25 and thebottom of flow channel 30 in FIG. 10.

Another advantage of having a curved upper flow channel surface atmembrane 25A is that the membrane can more readily conform to the shapeand volume of the flow channel in response to actuation. Specifically,where a rectangular flow channel is employed, the entire perimeter (2×flow channel height, plus the flow channel width) must be forced intothe flow channel. However where an arched flow channel is used, asmaller perimeter of material (only the semi-circular arched portion)must be forced into the channel. In this manner, the membrane requiresless change in perimeter for actuation and is therefore more responsiveto an applied actuation force to block the flow channel

In an alternate aspect, (not illustrated), the bottom of flow channel 30is rounded such that its curved surface mates with the curved upper wall25A as seen in FIG. 20 described above.

In summary, the actual conformational change experienced by the membraneupon actuation will depend upon the configuration of the particularelastomeric structure. Specifically, the conformational change willdepend upon the length, width, and thickness profile of the membrane,its attachment to the remainder of the structure, and the height, width,and shape of the flow and control channels and the material propertiesof the elastomer used. The conformational change may also depend uponthe method of actuation, as actuation of the membrane in response to anapplied pressure will vary somewhat from actuation in response to amagnetic or electrostatic force.

Moreover, the desired conformational change in the membrane will alsovary depending upon the particular application for the elastomericstructure. In the simplest embodiments described above, the valve mayeither be open or closed, with metering to control the degree of closureof the valve. In other embodiments however, it may be desirable to alterthe shape of the membrane and/or the flow channel in order to achievemore complex flow regulation. For instance, the flow channel could beprovided with raised protrusions beneath the membrane portion, such thatupon actuation the membrane shuts off only a percentage of the flowthrough the flow channel, with the percentage of flow blockedinsensitive to the applied actuation force.

Many membrane thickness profiles and flow channel cross-sections arecontemplated by the present invention, including rectangular,trapezoidal, circular, ellipsoidal, parabolic, hyperbolic, andpolygonal, as well as sections of the above shapes. More complexcross-sectional shapes, such as the embodiment with protrusionsdiscussed immediately above or an embodiment having concavities in theflow channel, are also contemplated by the present invention.

In addition, while the invention is described primarily above inconjunction with an embodiment wherein the walls and ceiling of the flowchannel are formed from elastomer, and the floor of the channel isformed from an underlying substrate, the present invention is notlimited to this particular orientation. Walls and floors of channelscould also be formed in the underlying substrate, with only the ceilingof the flow channel constructed from elastomer. This elastomer flowchannel ceiling would project downward into the channel in response toan applied actuation force, thereby controlling the flow of materialthrough the flow channel. In general, monolithic elastomer structures asdescribed elsewhere in the instant application are preferred formicrofluidic applications. However, it may be useful to employ channelsformed in the substrate where such an arrangement provides advantages.For instance, a substrate including optical waveguides could beconstructed so that the optical waveguides direct light specifically tothe side of a microfluidic channel.

7. Alternate Valve Actuation Techniques

In addition to pressure based actuation systems described above,optional electrostatic and magnetic actuation systems are alsocontemplated, as follows.

Electrostatic actuation can be accomplished by forming oppositelycharged electrodes (which will tend to attract one another when avoltage differential is applied to them) directly into the monolithicelastomeric structure. For example, referring to FIG. 7B, an optionalfirst electrode 70 (shown in phantom) can be positioned on (or in)membrane 25 and an optional second electrode 72 (also shown in phantom)can be positioned on (or in) planar substrate 14. When electrodes 70 and72 are charged with opposite polarities, an attractive force between thetwo electrodes will cause membrane 25 to deflect downwardly, therebyclosing the “valve” (i.e.: closing flow channel 30).

For the membrane electrode to be sufficiently conductive to supportelectrostatic actuation, but not so mechanically stiff so as to impedethe valve's motion, a sufficiently flexible electrode must be providedin or over membrane 25. Such an electrode may be provided by a thinmetallization layer, doping the polymer with conductive material, ormaking the surface layer out of a conductive material.

In an exemplary aspect, the electrode present at the deflecting membranecan be provided by a thin metallization layer which can be provided, forexample, by sputtering a thin layer of metal such as 20 nm of gold. Inaddition to the formation of a metallized membrane by sputtering, othermetallization approaches such as chemical epitaxy, evaporation,electroplating, and electroless plating are also available. Physicaltransfer of a metal layer to the surface of the elastomer is alsoavailable, for example by evaporating a metal onto a flat substrate towhich it adheres poorly, and then placing the elastomer onto the metaland peeling the metal off of the substrate.

A conductive electrode 70 may also be formed by depositing carbon black(i.e. Cabot Vulcan XC72R) on the elastomer surface, either by wiping onthe dry powder or by exposing the elastomer to a suspension of carbonblack in a solvent which causes swelling of the elastomer, (such as achlorinated solvent in the case of PDMS). Alternatively, the electrode70 may be formed by constructing the entire layer 20 out of elastomerdoped with conductive material (i.e. carbon black or finely dividedmetal particles). Yet further alternatively, the electrode may be formedby electrostatic deposition, or by a chemical reaction that producescarbon. In experiments conducted by the present inventors, conductivitywas shown to increase with carbon black concentration from 5.6×10⁻¹⁶ toabout 5×10⁻³ (Ω-cm)⁻¹. The lower electrode 72, which is not required tomove, may be either a compliant electrode as described above, or aconventional electrode such as evaporated gold, a metal plate, or adoped semiconductor electrode.

Magnetic actuation of the flow channels can be achieved by fabricatingthe membrane separating the flow channels with a magneticallypolarizable material such as iron, or a permanently magnetized materialsuch as polarized NdFeB. In experiments conducted by the presentinventors, magnetic silicone was created by the addition of iron powder(about 1 um particle size), up to 20% iron by weight.

Where the membrane is fabricated with a magnetically polarizablematerial, the membrane can be actuated by attraction in response to anapplied magnetic field Where the membrane is fabricated with a materialcapable of maintaining permanent magnetization, the material can firstbe magnetized by exposure to a sufficiently high magnetic field, andthen actuated either by attraction or repulsion in response to thepolarity of an applied in homogenous magnetic field.

The magnetic field causing actuation of the membrane can be generated ina variety of ways. In one embodiment, the magnetic field is generated byan extremely small inductive coil formed in or proximate to theelastomer membrane. The actuation effect of such a magnetic coil wouldbe localized, allowing actuation of individual pump and/or valvestructures. Alternatively, the magnetic field could be generated by alarger, more powerful source, in which case actuation would be globaland would actuate multiple pump and/or valve structures at one time.

It is also possible to actuate the device by causing a fluid flow in thecontrol channel based upon the application of thermal energy, either bythermal expansion or by production of gas from liquid. For example, inone alternative embodiment in accordance with the present invention, apocket of fluid (e.g. in a fluid-filled control channel) is positionedover the flow channel. Fluid in the pocket can be in communication witha temperature variation system, for example a heater. Thermal expansionof the fluid, or conversion of material from the liquid to the gasphase, could result in an increase in pressure, closing the adjacentflow channel. Subsequent cooling of the fluid would relieve pressure andpermit the flow channel to open.

8. Networked Systems

FIGS. 12A and 12B show a views of a single on/off valve, identical tothe systems set forth above, (for example in FIG. 7A). FIGS. 13A and 13Bshows a peristaltic pumping system comprised of a plurality of thesingle addressable on/off valves as seen in FIG. 12, but networkedtogether. FIG. 14 is a graph showing experimentally achieved pumpingrates vs. frequency for the peristaltic pumping system of FIG. 13. FIGS.15A and 15B show a schematic view of a plurality of flow channels whichare controllable by a single control line. This system is also comprisedof a plurality of the single addressable on/off valves of FIG. 12,multiplexed together, but in a different arrangement than that of FIG.12. FIG. 16 is a schematic illustration of a multiplexing system adaptedto permit fluid flow through selected channels, comprised of a pluralityof the single on/off valves of FIG. 12, joined or networked together.

Referring first to FIGS. 12A and 12B, a schematic of flow channels 30and 32 is shown. Flow channel 30 preferably has a fluid (or gas) flow Fpassing therethrough. Flow channel 32, (which crosses over flow channel30, as was already explained herein), is pressurized such that membrane25 separating the flow channels may be depressed into the path of flowchannel 30, shutting off the passage of flow F therethrough, as has beenexplained. As such, “flow channel” 32 can also be referred to as a“control line” which actuates a single valve in flow channel 30. InFIGS. 12 to 15, a plurality of such addressable valves are joined ornetworked together in various arrangements to produce pumps, capable ofperistaltic pumping, and other fluidic logic applications.

Referring to FIGS. 13A and 13B, a system for peristaltic pumping isprovided, as follows. A flow channel 30 has a plurality of generallyparallel flow channels (i.e.: control lines) 32A, 32B and 32C passingthereover. By pressurizing control line 32A, flow F through flow channel30 is shut off under membrane 25A at the intersection of control line32A and flow channel 30. Similarly, (but not shown), by pressurizingcontrol line 32B, flow F through flow channel 30 is shut off undermembrane 25B at the intersection of control line 32B and flow channel30, etc.

Each of control lines 32A, 32B, and 32C is separately addressable.Therefore, peristalsis may be actuated by the pattern of actuating 32Aand 32C together, followed by 32A, followed by 32A and 32B together,followed by 32B, followed by 32B and C together, etc. This correspondsto a successive “101, 100, 110, 010, 011, 001” pattern, where “0”indicates “valve open” and “1” indicates “valve closed.” Thisperistaltic pattern is also known as a 120° pattern (referring to thephase angle of actuation between three valves). Other peristalticpatterns are equally possible, including 60° and 90° patterns.

In experiments performed by the inventors, a pumping rate of 2.35 nL/swas measured by measuring the distance traveled by a column of water inthin (0.5 mm i.d.) tubing; with 100×100×10 μm valves under an actuationpressure of 40 kPa. The pumping rate increased with actuation frequencyuntil approximately 75 Hz, and then was nearly constant until above 200Hz. The valves and pumps are also quite durable and the elastomermembrane, control channels, or bond have never been observed to fail. Inexperiments performed by the inventors, none of the valves in theperistaltic pump described herein show any sign of wear or fatigue aftermore than 4 million actuations. In addition to their durability, theyare also gentle. A solution of E. coli pumped through a channel andtested for viability showed a 94% survival rate.

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for the peristaltic pumping system of FIG. 13.

FIGS. 15A and 15B illustrates another way of assembling a plurality ofthe addressable valves of FIG. 12. Specifically, a plurality of parallelflow channels 30A, 30B, and 30C are provided. Flow channel (i.e.:control line) 32 passes thereover across flow channels 30A, 30B, and30C. Pressurization of control line 32 simultaneously shuts off flowsF1, F2 and F3 by depressing membranes 25A, 25B, and 25C located at theintersections of control line 32 and flow channels 30A, 30B, and 30C.

FIG. 16 is a schematic illustration of a multiplexing system adapted toselectively permit fluid to flow through selected channels, as follows.The downward deflection of membranes separating the respective flowchannels from a control line passing thereabove (for example, membranes25A, 25B, and 25C in FIGS. 15A and 15B) depends strongly upon themembrane dimensions. Accordingly, by varying the widths of flow channelcontrol line 32 in FIGS. 15A and 15B, it is possible to have a controlline pass over multiple flow channels, yet only actuate (i.e.: seal)desired flow channels. FIG. 16 illustrates a schematic of such a system,as follows.

A plurality of parallel flow channels 30A, 30B, 30C, 30D, 30E and 30Fare positioned under a plurality of parallel control lines 32A, 32B,32C, 32D, 32E and 32F. Control channels 32A, 32B, 32C, 32D, 32E and 32Fare adapted to shut off fluid flows F1, F2, F3, F4, F5 and F6 passingthrough parallel flow channels 30A, 30B, 30C, 30D, 30E and 30F using anyof the valving systems described above, with the following modification.

Each of control lines 32A, 32B, 32C, 32D, 32E and 32F have both wide andnarrow portions. For example, control line 32A is wide in locationsdisposed over flow channels 30A, 30C and 30E. Similarly, control line32B is wide in locations disposed over flow channels 30B, 30D and 30F,and control line 32C is wide in locations disposed over flow channels30A, 30B, 30E and 30F.

At the locations where the respective control line is wide, itspressurization will cause the membrane (25) separating the flow channeland the control line to depress significantly into the flow channel,thereby blocking the flow passage therethrough. Conversely, in thelocations where the respective control line is narrow, membrane (25)will also be narrow. Accordingly, the same degree of pressurization willnot result in membrane (25) becoming depressed into the flow channel(30). Therefore, fluid passage thereunder will not be blocked.

For example, when control line 32A is pressurized, it will block flowsF1, F3 and F5 in flow channels 30A, 30C and 30E. Similarly, when controlline 32C is pressurized, it will block flows F1, F2, F5 and F6 in flowchannels 30A, 30B, 30E and 30F. As can be appreciated, more than onecontrol line can be actuated at the same time. For example, controllines 32A and 32C can be pressurized simultaneously to block all fluidflow except F4 (with 32A blocking F1, F3 and F5; and 32C blocking F1,F2, F5 and F6).

By selectively pressurizing different control lines (32) both togetherand in various sequences, a great degree of fluid flow control can beachieved. Moreover, by extending the present system to more than sixparallel flow channels (30) and more than four parallel control lines(32), and by varying the positioning of the wide and narrow regions ofthe control lines, very complex fluid flow control systems may befabricated. A property of such systems is that it is possible to turn onany one flow channel out of n flow channels with only 2(log 2n) controllines.

9. Selectively Addressable Reaction Chambers Along Flow Lines

In a further embodiment of the invention, illustrated in FIGS. 17A, 17B,17C and 17D, a system for selectively directing fluid flow into one moreof a plurality of reaction chambers disposed along a flow line isprovided.

FIG. 17A shows a top view of a flow channel 30 having a plurality ofreaction chambers 80A and 80B disposed therealong. Preferably flowchannel 30 and reaction chambers 80A and 80B are formed together asrecesses into the bottom surface of a first layer 100 of elastomer.

FIG. 17B shows a bottom plan view of another elastomeric layer 110 withtwo control lines 32A and 32B each being generally narrow, but havingwide extending portions 33A and 33B formed as recesses therein.

As seen in the exploded view of FIG. 17C, and assembled view of FIG.17D, elastomeric layer 110 is placed over elastomeric layer 100. Layers100 and 110 are then bonded together, and the integrated system operatesto selectively direct fluid flow F (through flow channel 30) into eitheror both of reaction chambers 80A and 80B, as follows. Pressurization ofcontrol line 32A will cause the membrane 25 (i.e.: the thin portion ofelastomer layer 100 located below extending portion 33A and over regions82A of reaction chamber 80A) to become depressed, thereby shutting offfluid flow passage in regions 82A, effectively sealing reaction chamber80 from flow channel 30. As can also be seen, extending portion 33A iswider than the remainder of control line 32A. As such, pressurization ofcontrol line 32A will not result in control line 32A sealing flowchannel 30.

As can be appreciated, either or both of control lines 32A and 32B canbe actuated at once. When both control lines 32A and 32B are pressurizedtogether, sample flow in flow channel 30 will enter neither of reactionchambers 80A or 80B.

The concept of selectably controlling fluid introduction into variousaddressable reaction chambers disposed along a flow line (FIGS. 17A-D)can be combined with concept of selectably controlling fluid flowthrough one or more of a plurality of parallel flow lines (FIG. 16) toyield a system in which a fluid sample or samples can be can be sent toany particular reaction chamber in an array of reaction chambers. Anexample of such a system is provided in FIG. 18, in which parallelcontrol channels 32A, 32B and 32C with extending portions 34 (all shownin phantom) selectively direct fluid flows F1 and F2 into any of thearray of reaction wells 80A, 80B, 80C or 80D as explained above; whilepressurization of control lines 32C and 32D selectively shuts off flowsF2 and F1, respectively.

In yet another novel embodiment, fluid passage between parallel flowchannels is possible. Referring to FIG. 19, either or both of controllines 32A or 32D can be depressurized such that fluid flow throughlateral passageways 35 (between parallel flow channels 30A and 30B) ispermitted. In this aspect of the invention, pressurization of controllines 32C and 32D would shut flow channel 30A between 35A and 35B, andwould also shut lateral passageways 35B. As such, flow entering as flowF1 would sequentially travel through 30A, 35A and leave 30B as flow F4.

10. Switchable Flow Arrays

In yet another novel embodiment, fluid passage can be selectivelydirected to flow in either of two perpendicular directions. An exampleof such a “switchable flow array” system is provided in FIGS. 20A to20D. FIG. 20A shows a bottom view of a first layer of elastomer 90, (orany other suitable substrate), having a bottom surface with a pattern ofrecesses forming a flow channel grid defined by an array of solid posts92, each having flow channels passing therearound.

In preferred aspects, an additional layer of elastomer is bound to thetop surface of layer 90 such that fluid flow can be selectively directedto move either in direction F1, or perpendicular direction F2. FIG. 20is a bottom view of the bottom surface of the second layer of elastomer95 showing recesses formed in the shape of alternating “vertical”control lines 96 and “horizontal” control lines 94. “Vertical” controllines 96 have the same width therealong, whereas “horizontal” controllines 94 have alternating wide and narrow portions, as shown.

Elastomeric layer 95 is positioned over top of elastomeric layer 90 suchthat “vertical” control lines 96 are positioned over posts 92 as shownin FIG. 20C and “horizontal” control lines 94 are positioned with theirwide portions between posts 92, as shown in FIG. 20D.

As can be seen in FIG. 20C, when “vertical” control lines 96 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 98 will be deflected downwardly over the array of flow channelssuch that flow in only able to pass in flow direction F2 (i.e.:vertically), as shown.

As can be seen in FIG. 20D, when “horizontal” control lines 94 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 99 will be deflected downwardly over the array of flow channels,(but only in the regions where they are widest), such that flow in onlyable to pass in flow direction F1 (i.e.: horizontally), as shown.

The design illustrated in FIG. 20 allows a switchable flow array to beconstructed from only two elastomeric layers, with no vertical viaspassing between control lines in different elastomeric layers required.If all vertical flow control lines 94 are connected, they may bepressurized from one input. The same is true for all horizontal flowcontrol lines 96.

11. Normally-Closed Valve Structure

FIGS. 7B and 7H above depict a valve structure in which the elastomericmembrane is moveable from a first relaxed position to a second actuatedposition in which the flow channel is blocked. However, the presentinvention is not limited to this particular valve configuration.

FIGS. 21A-21J show a variety of views of a normally-closed valvestructure in which the elastomeric membrane is moveable from a firstrelaxed position blocking a flow channel, to a second actuated positionin which the flow channel is open, utilizing a negative controlpressure.

FIG. 21A shows a plan view, and FIG. 21B shows a cross sectional viewalong line 42B-42B′, of normally-closed valve 4200 in an unactuatedstate. Flow channel 4202 and control channel 4204 are formed inelastomeric block 4206 overlying substrate 4205. Flow channel 4202includes a first portion 4202 a and a second portion 4202 b separated byseparating portion 4208. Control channel 4204 overlies separatingportion 4208. As shown in FIG. 42B, in its relaxed, unactuated position,separating portion 4008 remains positioned between flow channel portions4202 a and 4202 b, interrupting flow channel 4202.

FIG. 21C shows a cross-sectional view of valve 4200 wherein separatingportion 4208 is in an actuated position. When the pressure withincontrol channel 4204 is reduced to below the pressure in the flowchannel (for example by vacuum pump), separating portion 4208experiences an actuating force drawing it into control channel 4204. Asa result of this actuation force membrane 4208 projects into controlchannel 4204, thereby removing the obstacle to a flow of materialthrough flow channel 4202 and creating a passageway 4203. Upon elevationof pressure within control channel 4204, separating portion 4208 willassume its natural position, relaxing back into and obstructing flowchannel 4202.

The behavior of the membrane in response to an actuation force may bechanged by varying the width of the overlying control channel.Accordingly, FIGS. 21D-42H show plan and cross-sectional views of analternative embodiment of a normally-closed valve 4201 in which controlchannel 4207 is substantially wider than separating portion 4208. Asshown in cross-sectional views FIG. 21E-F along line 42E-42E′ of FIG.21D, because a larger area of elastomeric material is required to bemoved during actuation, the actuation force necessary to be applied isreduced.

FIGS. 21G and H show a cross-sectional views along line 40G-40G′ of FIG.21D. In comparison with the unactuated valve configuration shown in FIG.21G, FIG. 21H shows that reduced pressure within wider control channel4207 may under certain circumstances have the unwanted effect of pullingunderlying elastomer 4206 away from substrate 4205, thereby creatingundesirable void 4212.

Accordingly, FIG. 21I shows a plan view, and FIG. 21J shows across-sectional view along line 21J-21J′ of FIG. 21I, of valve structure4220 which avoids this problem by featuring control line 4204 with aminimum width except in segment 4204 a overlapping separating portion4208. As shown in FIG. 21J, even under actuated conditions the narrowercross-section of control channel 4204 reduces the attractive force onthe underlying elastomer material 4206, thereby preventing thiselastomer material from being drawn away from substrate 4205 andcreating an undesirable void.

While a normally-closed valve structure actuated in response to pressureis shown in FIGS. 21A-21J, a normally-closed valve in accordance withthe present invention is not limited to this configuration. For example,the separating portion obstructing the flow channel could alternativelybe manipulated by electric or magnetic fields, as described extensivelyabove.

12. Side-Actuated Valve

While the above description has focused upon microfabricated elastomericvalve structures in which a control channel is positioned above andseparated by an intervening elastomeric membrane from an underlying flowchannel, the present invention is not limited to this configuration.FIGS. 22A and 22B show plan views of one embodiment of a side-actuatedvalve structure in accordance with one embodiment of the presentinvention.

FIG. 22A shows side-actuated valve structure 4800 in an unactuatedposition. Flow channel 4802 is formed in elastomeric layer 4804. Controlchannel 4806 abutting flow channel 4802 is also formed in elastomericlayer 4804. Control channel 4806 is separated from flow channel 4802 byelastomeric membrane portion 4808. A second elastomeric layer (notshown) is bonded over bottom elastomeric layer 4804 to enclose flowchannel 4802 and control channel 4806.

FIG. 22B shows side-actuated valve structure 4800 in an actuatedposition. In response to a build up of pressure within control channel4806, membrane 4808 deforms into flow channel 4802, blocking flowchannel 4802. Upon release of pressure within control channel 4806,membrane 4808 would relax back into control channel 4806 and open flowchannel 4802.

While a side-actuated valve structure actuated in response to pressureis shown in FIGS. 22A and 22B, a side-actuated valve in accordance withthe present invention is not limited to this configuration. For example,the elastomeric membrane portion located between the abutting flow andcontrol channels could alternatively be manipulated by electric ormagnetic fields, as described extensively above.

13. Composite Structures

Microfabricated elastomeric structures of the present invention may becombined with non-elastomeric materials to create composite structures.FIG. 23 shows a cross-sectional view of one embodiment of a compositestructure in accordance with the present invention. FIG. 23 showscomposite valve structure 5700 including first, thin elastomer layer5702 overlying semiconductor-type substrate 5704 having channel 5706formed therein. Second, thicker elastomer layer 5708 overlies firstelastomer layer 5702. Actuation of first elastomer layer 5702 to driveit into channel 5706, will cause composite structure 5700 to operate asa valve.

FIG. 24 shows a cross-sectional view of a variation on this theme,wherein thin elastomer layer 5802 is sandwiched between two hard,semiconductor substrates 5804 and 5806, with lower substrate 5804featuring channel 5808. Again, actuation of thin elastomer layer 5802 todrive it into channel 5808 will cause composite structure 5810 tooperate as a valve.

The structures shown in FIG. 23 or 24 may be fabricated utilizing eitherthe multilayer soft lithography or encapsulation techniques describedabove. In the multilayer soft lithography method, the elastomer layer(s)would be formed and then placed over the semiconductor substrate bearingthe channel. In the encapsulation method, the channel would be firstformed in the semiconductor substrate, and then the channel would befilled with a sacrificial material such as photoresist. The elastomerwould then be formed in place over the substrate, with removal of thesacrificial material producing the channel overlaid by the elastomermembrane. As is discussed in detail below in connection with bonding ofelastomer to other types of materials, the encapsulation approach mayresult in a stronger seal between the elastomer membrane component andthe underlying nonelastomer substrate component.

As shown in FIGS. 23 and 24, a composite structure in accordance withembodiments of the present invention may include a hard substrate thatbears a passive feature such as a channels. However, the presentinvention is not limited to this approach, and the underlying hardsubstrate may bear active features that interact with an elastomercomponent bearing a recess. This is shown in FIG. 25, wherein compositestructure 5900 includes elastomer component 5902 containing recess 5904having walls 5906 and ceiling 5908. Ceiling 5908 forms flexible membraneportion 5909. Elastomer component 5902 is sealed against substantiallyplanar nonelastomeric component 5910 that includes active device 5912.Active device 5912 may interact with material present in recess 5904and/or flexible membrane portion 5909.

Many Types of active structures may be present in the nonelastomersubstrate. Active structures that could be present in an underlying hardsubstrate include, but are not limited to, resistors, capacitors,photodiodes, transistors, chemical field effect transistors (chemFET's), amperometric/coulometric electrochemical sensors, fiber optics,fiber optic interconnects, light emitting diodes, laser diodes, verticalcavity surface emitting lasers (VCSEL's), micromirrors, accelerometers,pressure sensors, flow sensors, CMOS imaging arrays, CCD cameras,electronic logic, microprocessors, thermistors, Peltier coolers,waveguides, resistive heaters, chemical sensors, strain gauges,inductors, actuators (including electrostatic, magnetic,electromagnetic, bimetallic, piezoelectric, shape-memory-alloy based,and others), coils, magnets, electromagnets, magnetic sensors (such asthose used in hard drives, superconducting quantum interference devices(SQUIDS) and other types), radio frequency sources and receivers,microwave frequency sources and receivers, sources and receivers forother regions of the electromagnetic spectrum, radioactive particlecounters, and electrometers.

As is well known in the art, a vast variety of technologies can beutilized to fabricate active features in semiconductor and other typesof hard substrates, including but not limited printed circuit board(PCB) technology, CMOS, surface micromachining, bulk micromachining,printable polymer electronics, and TFT and otheramorphous/polycrystalline techniques as are employed to fabricate laptopand flat screen displays.

A variety of approaches can be employed to seal the elastomericstructure against the nonelastomeric substrate, ranging from thecreation of a Van der Waals bond between the elastomeric andnonelastomeric components, to creation of covalent or ionic bondsbetween the elastomeric and nonelastomeric components of the compositestructure. Example approaches to sealing the components together arediscussed below, approximately in order of increasing strength.

A first approach is to rely upon the simple hermetic seal resulting fromVan der Waals bonds formed when a substantially planar elastomer layeris placed into contact with a substantially planar layer of a harder,non-elastomer material. In one embodiment, bonding of RTV elastomer to aglass substrate created a composite structure capable of withstanding upto about 3-4 psi of pressure. This may be sufficient for many potentialapplications.

A second approach is to utilize a liquid layer to assist in bonding. Oneexample of this involves bonding elastomer to a hard glass substrate,wherein a weakly acidic solution (5 μl HCl in H₂0, pH 2) was applied toa glass substrate. The elastomer component was then placed into contactwith the glass substrate, and the composite structure baked at 37° C. toremove the water. This resulted in a bond between elastomer andnon-elastomer able to withstand a pressure of about 20 psi. In thiscase, the acid may neutralize silanol groups present on the glasssurface, permitting the elastomer and non-elastomer to enter into goodVan der Waals contact with each other.

Exposure to ethanol can also cause device components to adhere together.In one embodiment, an RTV elastomer material and a glass substrate werewashed with ethanol and then dried under Nitrogen. The RTV elastomer wasthen placed into contact with the glass and the combination baked for 3hours at 80° C. Optionally, the RTV may also be exposed to a vacuum toremove any air bubbles trapped between the slide and the RTV. Thestrength of the adhesion between elastomer and glass using this methodhas withstood pressures in excess of 35 psi. The adhesion created usingthis method is not permanent, and the elastomer may be peeled off of theglass, washed, and resealed against the glass. This ethanol washingapproach can also be employed used to cause successive layers ofelastomer to bond together with sufficient strength to resist a pressureof 30 psi. In alternative embodiments, chemicals such as other alcoholsor diols could be used to promote adhesion between layers.

An embodiment of a method of promoting adhesion between layers of amicrofabricated structure in accordance with the present inventioncomprises exposing a surface of a first component layer to a chemical,exposing a surface of a second component layer to the chemical, andplacing the surface of the first component layer into contact with thesurface of the second elastomer layer.

A third approach is to create a covalent chemical bond between theelastomer component and functional groups introduced onto the surface ofa nonelastomer component. Examples of derivitization of a nonelastomersubstrate surface to produce such functional groups include exposing aglass substrate to agents such as vinyl silane or aminopropyltriethoxysilane (APTES), which may be useful to allow bonding of the glass tosilicone elastomer and polyurethane elastomer materials, respectively.

A fourth approach is to create a covalent chemical bond between theelastomer component and a functional group native to the surface of thenonelastomer component. For example, RTV elastomer can be created withan excess of vinyl groups on its surface. These vinyl groups can becaused to react with corresponding functional groups present on theexterior of a hard substrate material, for example the Si—H bondsprevalent on the surface of a single crystal silicon substrate afterremoval of native oxide by etching. In this example, the strength of thebond created between the elastomer component and the nonelastomercomponent has been observed to exceed the materials strength of theelastomer components.

14. Cell Pen/Cell Cage

In yet a further application of the present invention, an elastomericstructure can be utilized to manipulate organisms or other biologicalmaterial. FIGS. 26A-26D show plan views of one embodiment of a cell penstructure in accordance with the present invention.

Cell pen array 4400 features an array of orthogonally-oriented flowchannels 4402, with an enlarged “pen” structure 4404 at the intersectionof alternating flow channels. Valve 4406 is positioned at the entranceand exit of each pen structure 4404. Peristaltic pump structures 4408are positioned on each horizontal flow channel and on the vertical flowchannels lacking a cell pen structure.

Cell pen array 4400 of FIG. 26A has been loaded with cells A-H that havebeen previously sorted. FIGS. 26B-26C show the accessing and removal ofindividually stored cell C by 1) opening valves 4406 on either side ofadjacent pens 4404 a and 4404 b, 2) pumping horizontal flow channel 4402a to displace cells C and G, and then 3) pumping vertical flow channel4402 b to remove cell C. FIG. 26D shows that second cell G is moved backinto its prior position in cell pen array 4400 by reversing thedirection of liquid flow through horizontal flow channel 4402 a.

The cell pen array 4404 described above is capable of storing materialswithin a selected, addressable position for ready access. However,living organisms such as cells may require a continuous intake of foodsand expulsion of wastes in order to remain viable. Accordingly, FIGS.27A and 27B show plan and cross-sectional views (along line 45B-45B′)respectively, of one embodiment of a cell cage structure in accordancewith the present invention.

Cell cage 4500 is formed as an enlarged portion 4500 a of a flow channel4501 in an elastomeric block 4503 in contact with substrate 4505. Cellcage 4500 is similar to an individual cell pen as described above inFIGS. 26A-26D, except that ends 4500 b and 4500 c of cell cage 4500 donot completely enclose interior region 4500 a. Rather, ends 4500 a and4500 b of cage 4500 are formed by a plurality of retractable pillars4502. Pillars 4502 may be part of a membrane structure of anormally-closed valve structure as described extensively above inconnection with FIGS. 21A-21J.

Specifically, control channel 4504 overlies pillars 4502. When thepressure in control channel 4504 is reduced, elastomeric pillars 4502are drawn upward into control channel 4504, thereby opening end 4500 bof cell cage 4500 and permitting a cell to enter. Upon elevation ofpressure in control channel 4504, pillars 4502 relax downward againstsubstrate 4505 and prevent a cell from exiting cage 4500.

Elastomeric pillars 4502 are of a sufficient size and number to preventmovement of a cell out of cage 4500, but also include gaps 4508 whichallow the flow of nutrients into cage interior 4500 a in order tosustain cell(s) stored therein. Pillars 4502 on opposite end 4500 c aresimilarly configured beneath second control channel 4506 to permitopening of the cage and removal of the cell as desired.

The cross-flow channel architecture illustrated shown in FIGS. 26A-26Dcan be used to perform functions other than the cell pen just described.For example, the cross-flow channel architecture can be utilized inmixing applications.

This is shown in FIGS. 28A-B, which illustrate a plan view of mixingsteps performed by a microfabricated structure in accordance anotherembodiment of the present invention. Specifically, portion 7400 of amicrofabricated mixing structure comprises first flow channel 7402orthogonal to and intersecting with second flow channel 7404. Controlchannels 7406 overlie flow channels 7402 and 7404 and form valve pairs7408 a-b and 7408 c-d that surround each intersection 7412.

As shown in FIG. 28A, valve pair 7408 a-b is initially opened whilevalve pair 7408 c-d is closed, and fluid sample 7410 is flowed tointersection 7412 through flow channel 7402. Valve pair 7408 c-d is thenactuated, trapping fluid sample 7410 at intersection 7412.

Next, as shown in FIG. 28B, valve pairs 7408 a-b and 7408 c-d areopened, such that fluid sample 7410 is injected from intersection 7412into flow channel 7404 bearing a cross-flow of fluid. The process shownin FIGS. 28A-B can be repeated to accurately dispense any number offluid samples down cross-flow channel 7404.

While the embodiment shown and described above in connection with FIGS.28A-28B utilizes linked valve pairs on opposite sides of the flowchannel intersections, this is not required by the present invention.Other configurations, including linking of adjacent valves of anintersection, or independent actuation of each valve surrounding anintersection, are possible to provide the desired flow characteristics.With the independent valve actuation approach however, it should berecognized that separate control structures would be utilized for eachvalve, complicating device layout.

15. Metering by Volume Exclusion

Many high throughput screening and diagnostic applications call foraccurate combination and of different reagents in a reaction chamber.Given that it is frequently necessary to prime the channels of amicrofluidic device in order to ensure fluid flow, it may be difficultto ensure mixed solutions do not become diluted or contaminated by thecontents of the reaction chamber prior to sample introduction.

Volume exclusion is one technique enabling precise metering of theintroduction of fluids into a reaction chamber. In this approach, areaction chamber may be completely or partially emptied prior to sampleinjection. This method reduces contamination from residual contents ofthe chamber contents, and may be used to accurately meter theintroduction of solutions in a reaction chamber.

Specifically, FIGS. 29A-29D show cross-sectional views of a reactionchamber in which volume exclusion is employed to meter reactants. FIG.29A shows a cross-sectional view of portion 6300 of a microfluidicdevice comprising first elastomer layer 6302 overlying second elastomerlayer 6304. First elastomer layer 6302 includes control chamber 6306 influid communication with a control channel (not shown). Control chamber6306 overlies and is separated from dead-end reaction chamber 6308 ofsecond elastomer layer 6304 by membrane 6310. Second elastomer layer6304 further comprises flow channel 6312 leading to dead-end reactionchamber 6308.

FIG. 29B shows the result of a pressure increase within control chamber6306. Specifically, increased control chamber pressure causes membrane6310 to flex downward into reaction chamber 6308, reducing by volume Vthe effective volume of reaction chamber 6308. This in turn excludes anequivalent volume V of reactant from reaction chamber 6308, such thatvolume V of first reactant X is output from flow channel 6312. The exactcorrelation between a pressure increase in control chamber 6306 and thevolume of material output from flow channel 6312 can be preciselycalibrated.

As shown in FIG. 29C, while elevated pressure is maintained withincontrol chamber 6306, volume V′ of second reactant Y is placed intocontact with flow channel 6312 and reaction chamber 6308.

In the next step shown in FIG. 29D, pressure within control chamber 6306is reduced to original levels. As a result, membrane 6310 relaxes andthe effective volume of reaction chamber 6308 increases. Volume V ofsecond reactant Y is sucked into the device. By varying the relativesize of the reaction and control chambers, it is possible to accuratelymix solutions at a specified relative concentration. It is worth notingthat the amount of the second reactant Y that is sucked into the deviceis solely dependent upon the excluded volume V, and is independent ofvolume V′ of Y made available at the opening of the flow channel.

While FIGS. 29A-29D show a simple embodiment of the present inventioninvolving a single reaction chamber, in more complex embodimentsparallel structures of hundreds or thousands of reaction chambers couldbe actuated by a pressure increase in a single control line.

Moreover, while the above description illustrates two reactants beingcombined at a relative concentration that fixed by the size of thecontrol and reaction chambers, a volume exclusion technique could beemployed to combine several reagents at variable concentrations in asingle reaction chamber. One possible approach is to use several,separately addressable control chambers above each reaction chamber. Anexample of this architecture would be to have ten separate control linesinstead of a single control chamber, allowing ten equivalent volumes tobe pushed out or sucked in.

Another possible approach would utilize a single control chamberoverlying the entire reaction chamber, with the effective volume of thereaction chamber modulated by varying the control chamber pressure. Inthis manner, analog control over the effective volume of the reactionchamber is possible. Analog volume control would in turn permit thecombination of many solutions reactants at arbitrary relativeconcentrations.

An embodiment of a method of metering a volume of fluid in accordancewith the present invention comprises providing a chamber having a volumein an elastomeric block separated from a control recess by anelastomeric membrane, and supplying a pressure to the control recesssuch that the membrane is deflected into the chamber and the volume isreduced by a calibrated amount, thereby excluding from the chamber thecalibrated volume of fluid.

16. Sorting

The present microfluidic pumps and valves can also e used in flowcytometers for cell sorting and DNA sizing. Sorting of objects basedupon size is extremely useful in many technical fields.

For example, many assays in biology require determination of the size ofmolecular-sized entities. Of particular importance is the measurement oflength distribution of DNA molecules in a heterogeneous solution. Thisis commonly done using gel electrophoresis, in which the molecules areseparated by their differing mobility in a gel matrix in an appliedelectric field, and their positions detected by absorption or emissionof radiation. The lengths of the DNA molecules are then inferred fromtheir mobility.

While powerful, electrophoretic methods pose disadvantages. For mediumto large DNA molecules, resolution, i.e. the minimum length differenceat which different molecular lengths may be distinguished, is limited toapproximately 10% of the total length. For extremely large DNAmolecules, the conventional sorting procedure is not workable. Moreover,gel electrophoresis is a relatively lengthy procedure, and may requireon the order of hours or days to perform.

The sorting of cellular-sized entities is also an important task.Conventional flow cell sorters are designed to have a flow chamber witha nozzle and are based on the principle of hydrodynamic focusing withsheath flow. Most conventional cell sorters combine the technology ofpiezo-electric drop generation and electrostatic deflection to achievedroplet generation and high sorting rates. However, this approach offerssome important disadvantages. One disadvantage is that the complexity,size, and expense of the sorting device requires that it be reusable inorder to be cost-effective. Reuse can in turn lead to problems withresidual materials causing contamination of samples and turbulent fluidflow.

Therefore, there is a need in the art for a simple, inexpensive, andeasily fabricated sorting device which relies upon the mechanicalcontrol of fluid flow rather than upon electrical interactions betweenthe particle and the solute.

FIG. 30 shows one embodiment of a sorting device in accordance with thepresent invention. Sorting device 3000 is formed from a switching valvestructure created from channels present in an elastomeric block.Specifically, flow channel 3002 is T-shaped, with stem 3002 a of flowchannel 3002 in fluid communication with sample reservoir 3004containing sortable entities 3006 of different types denoted by shape(square, circle, triangle, etc.). Left branch 3002 b of flow channel3002 is in fluid communication with waste reservoir 3008. Right branch3002 c of flow channel 3002 is in communication with collectionreservoir 3010.

Control channels 3012 a, 3012 b, and 3012 c overlie and are separatedfrom stem 3002 a of flow channel 3002 by elastomeric membrane portions3014 a, 3014 b, and 3014 c respectively. Together, stem 3002 a of flowchannel 3002 and control channels 3012 a, 3012 b, and 3012 c form firstperistaltic pump structure 3016 similar to that described at lengthabove.

Control channel 3012 d overlies and is separated from right branch 3002c of flow channel 3002 by elastomeric membrane portion 3014 d. Together,right branch 3002 c of flow channel 3002 and control channels 3012 dforms first valve structure 3018 a. Control channel 3012 e overlies andis separated from left branch 3002 c of flow channel 3002 by elastomericmembrane portion 3014 e. Together, left branch 3002 c of flow channel3002 and control channel 3012 e forms second valve structure 3018 b.

As shown in FIG. 30, stem 3602 a of flow channel 3002 narrowsconsiderably as it approaches detection window 3020 adjacent to thejunction of stem 3002 a, right branch 3002 b, and left branch 3002 c.Detection window 3020 is of sufficient width to allow for uniformillumination of this region. In one embodiment, the width of the stemnarrows from 100 μm to 5 μm at the detection window. The width of thestem at the detection window can be precisely formed using the softlithography or photoresist encapsulation fabrication techniquesdescribed extensively above, and will depend upon the nature and size ofthe entity to be sorted.

Operation of sorting device in accordance with one embodiment of thepresent invention is as follows.

The sample is diluted to a level such that only a single sortable entitywould be expected to be present in the detection window at any time.Peristaltic pump 3016 is activated by flowing a fluid through controlchannels 3012 a-c as described extensively above. In addition, secondvalve structure 3018 b is closed by flowing fluid through controlchannel 3012 e. As a result of the pumping action of peristaltic pump3016 and the blocking action of second valve 3018 b, fluid flows fromsample reservoir 3004 through detection window 3020 into waste reservoir3008. Because of the narrowing of stem 3004, sortable entities presentin sample reservoir 3004 are carried by this regular fluid flow, one ata time, through detection window 3020.

Radiation 3040 from source 3042 is introduced into detection window3020. This is possible due to the transmissive property of theelastomeric material. Absorption or emission of radiation 3040 bysortable entity 3006 is then detected by detector 3044.

If sortable entity 3006 a within detection window 3020 is intended to besegregated and collected by sorting device 3000, first valve 3018 a isactivated and second valve 3018 b is deactivated. This has the effect ofdrawing sortable entity 3006 a into collection reservoir 3010, and atthe same time transferring second sortable entity 3006 b into detectionwindow 3020. If second sortable entity 3002 b is also identified forcollection, peristaltic pump 3016 continues to flow fluid through rightbranch 3602 c of flow channel 3002 into collection reservoir 3610.However, if second entity 3006 b is not to be collected, first valve3018 a opens and second valve 3018 b closes, and first peristaltic pump3016 resumes pumping liquid through left branch 3002 b of flow channel3002 into waste reservoir 3008.

While one specific embodiment of a sorting device and a method foroperation thereof is described in connection with FIG. 30, the presentinvention is not limited to this embodiment. For example, fluid need notbe flowed through the flow channels using the peristaltic pumpstructure, but could instead be flowed under pressure with theelastomeric valves merely controlling the directionality of flow. In yetanother embodiment, a plurality of sorting structures could be assembledin series in order to perform successive sorting operations, with thewaste reservoir of FIG. 36 simply replaced by the stem of the nextsorting structure.

Moreover, a high throughput method of sorting could be employed, whereina continuous flow of fluid from the sample reservoir through the windowand junction into the waste reservoir is maintained until an entityintended for collection is detected in the window. Upon detection of anentity to be collected, the direction of fluid flow by the pumpstructure is temporarily reversed in order to transport the desiredparticle back through the junction into the collection reservoir. Inthis manner, the sorting device could utilize a higher flow rate, withthe ability to backtrack when a desired entity is detected. Such analternative high throughput sorting technique could be used when theentity to be collected is rare, and the need to backtrack infrequent.

Sorting in accordance with the present invention would avoid thedisadvantages of sorting utilizing conventional electrokinetic flow,such as bubble formation, a strong dependence of flow magnitude anddirection on the composition of the solution and surface chemistryeffects, a differential mobility of different chemical species, anddecreased viability of living organisms in the mobile medium.

II. Structures and Methods for Electronic Detection

As just described, embodiments of microfluidic structures in accordancewith the present invention may contain a variety of materials forsorting or other purposes. In order to detect the presence and identityof such a detectable entity at particular locations within themicrofluidic devices, changes in the electric or magnetic environmentmay be monitored.

For purposes of this application, the term detectable entity includesbut is not limited to, white and red blood cells, bacteria, viralparticles, macromolecules such as proteins (or protein subunits) andnucleic acids (or fragments thereof), polymer (e.g. latex) beads,inorganic microparticles or nanoparticles. A detectable entity can alsocomprise a change in the nature of the contents of the flow channel,such that one portion can be distinguished electrically from anotherportion.

Certain embodiments in accordance with the present invention rely uponelectronic-based detection schemes. Specifically, an electric field isapplied to a detection region within a microfabricated elastomericstructure. When a detectable entity enters the detection region, theelectrical properties of the detectable entity alter the electricalenvironment of the detection region. This changed electrical environmentmay alter the electric field, may generate an electrical currentresulting from application of the electric field, or may result in bothgenerating a current and altering the electric field. The presence ofthe detected entity may thus be revealed by monitoring the changedvoltages or currents.

Other embodiments in accordance with the present invention rely uponmagnetic-based detection schemes. Specifically, as a detectable entityenters a detection region, the magnetic properties of the detectableentity alter the magnetic environment of the detection region. Thischanged magnetic environment may induce a current in a nearby coil, ormay change resistance of a nearby magnetoresistive element. The presenceof the detected entity may thus be revealed by monitoring these voltagesand/or currents.

An embodiment of a method of detecting an entity in a microfabricatedelastomeric structure comprises defining a detection volume within themicrofabricated elastomeric structure, the detection volume receivingone detectable entity or ensemble of detected entities at a time. Anelectric field is applied to the detection volume, and a change in oneof an impedance and a current of the detection volume is measured as thedetectable entity traverses the detection volume.

1. Electronic Based Sensing

The electrical properties of the sample within the detection volume canbe described in many equivalent ways. In the instant application, theterm impedance is employed to encompass the concepts of both resistanceand capacitance.

In one embodiment of an electronic-based detection method in accordancewith the present invention, an electric field is applied transverse to adirection of flow of the detectable entity in the detection region, suchthat a change in impedance is observed as the detectable entity passesthrough the detection region. One prior application of a similartechnique is described by Sohn et al. in “Capacitance cytometry:Measuring biological cells one by one,” Proceedings of the NationalAcademy of Sciences, 97, 20, 10687-10690, (2000), incorporated byreference for all purposes herein. As with this reference, detectableentities in the form of cellular material may be detected by embodimentsin accordance with the present invention.

FIG. 31A shows a simplified plan view of the detection region of anembodiment of such a “transverse” electronic-based detection apparatusin accordance with the present invention. Specifically, detector 3100comprises flow channel 3102 formed in elastomeric layer 3104. Electrodes3108 and 3110 are disposed on opposite sides of flow channel 3102 todefine detection region 3106 sufficiently small as to allow only onedetectable entity 3111 to pass through at a time. Electrodes 3108 and3110 need not extend across the entire width or height of the flowchannel.

Operation of detector 3100 is as follows. A solution containing adetectable entity 3111 is flowed down flow channel 3102 and throughdetection region 3206. Electrodes 3108 and 3110 are placed into contactwith terminals 3112 a and 3112 b of AC power supply 3112. The frequencyof oscillation of the AC power supply relative to the flow rate would bechosen to be high enough as to allow detection of the passage of anentity entrained in the fluid flowing between the electrodes.

As a result of their orientation, electrodes 3108 and 3110 apply anelectric field in a direction transverse to the flow of materialsthrough detection region 3106. Application of a potential differenceacross electrodes 3108 and 3110 creates a capacitor structure having asplates electrodes 3108 and 3110, and having the contents of the flowchannel as a dielectric. The capacitance exhibited by thiselectrode/flow channel capacitor structure, and hence the voltagebetween the electrodes, remains constant as solute flows through.

However, as a detectable entity 3111 passes between electrodes 3108 and3110, the dielectric properties of the electrode/flow channel capacitorchange. Depending upon the electrical conductivity and permittivity ofdetectable entity 3111, capacitance (and also resistance) of theelectrode/flow channel capacitor structure may either rise or fall,resulting in a change in voltage across electrodes 3108 and 3110.

The change in voltage just described may be correlated with the presenceand/or identity of a detected entity within the flow channel. Bymonitoring voltage change signals over time, the number of detectableentities passing through the detection region can be counted. Inaddition, information on the size, orientation, and electricalproperties of the detectable entities may simultaneously be obtained.

The present invention is not limited to the embodiment shown in FIG. 31a. FIGS. 31B-D show perspective views of alternative embodiments ofdetection regions of a “transverse” electronic-based detectionapparatuses. The embodiment shown in FIG. 31B features opposedelectrodes positioned in the floor and ceiling of the flow channel. Theembodiment shown in FIG. 31C features opposed electrodes positioned in aplane of the floor of the flow channel.

While FIG. 31A shows the electrodes in communication with an AC voltagesource, in an alternative embodiment an electric field could be utilizedto pass a constant electrical current across the detection region. Whena detected entity is present therein, an increase in electric fieldrequired to maintain the original current level could be measured.

In an alternative embodiment of an electronic-based detection method inaccordance with the present invention, an electric field is appliedparallel to a direction of flow of the detectable entity in thedetection region, such that a change in impedance is observed as thedetectable entity passes through the detection region.

FIG. 32 shows a simplified plan view of the detection region of anembodiment of such a “longitudinal” electronic-based detection apparatusin accordance with the present invention. Detector 3200 operatesaccording to the well-known Coulter Principle.

Specifically, flow channel 3202 is formed in elastomeric layer 3104.Electrodes 3208 and 3210 are disposed adjacent to flow channel 3102 todefine detection region 3206 between them. Detection region 3206 issufficiently small as to allow only one detectable entity 3211 to passthrough at a time, and the flow channel is not necessarily straight, orof constant width, throughout the entire detection region. Electrodes3208 and 3210 need not extend across the entire width or height of theflow channel.

Operation of detector 3200 is as follows. A solution containing adetectable entity 3211 is flowed down flow channel 3202 and throughdetection region 3206. Electrodes 3208 and 3210 are placed into contactwith terminals 3212 a and 3212 b of AC power supply 3212. The frequencyof oscillation of the AC power supply relative to the flow rate would bechosen to be high enough as to allow detection of the passage of anentity entrained in the fluid flowing between the electrodes.

As a result of their orientation, electrodes 3208 and 3210 apply anelectric field in a direction parallel to the flow of materials throughdetection region 3206. Where only solvent is present in detection region3206, the potential difference across electrodes 3208 and 3210 givesrise to a current flow of a given magnitude. However, this current flowchanges when detectable entity 3211 enters detection region 3206.Specifically, the presence of detectable entity 3211 within detectionregion 3206 alters the continuous conductive path between electrodes3208 and 3210 through solute within detection region 3206. As a resultof this change in conductivity of the current path between electrodes3208 and 3210, the amount of current passing between the electrodeschanges.

Where the detectable entity exhibits an electrical conductivity that isgreater than the solute, the current passing between the electrodes mayincrease. Where the detectable entity exhibits an electricalconductivity that is less than the solute, the current passing betweenthe electrodes may decrease.

While FIG. 32 shows the electrodes in communication with an AC voltagesource, in an alternative embodiment an electric field could be utilizedto pass a constant electrical current along the detection region. When adetected entity is present therein, an increase in electric fieldrequired to maintain the original current level could be measured.

The changes in electrical properties just described may be correlatedwith the presence and identity of one or more detected entities withinthe constriction. By monitoring current between the electrodes, overtime the number of entities passing through the constriction can becounted. In addition, by correlating the magnitude of the change involtage or current with the characteristics of the detectable entity,information regarding the size, orientation, and electrical propertiesof the detectable entities may simultaneously be obtained. The size andorientation of the detected entities can often be obtained with minimalcomputation, as can the rate of flow through the detection region. Inaddition, electrical properties such as conductivity and permittivity ofthe detected entities influence the data, and can therefore be inferred.

The longitudinal sensing architecture described in FIG. 32 may beutilized to sense the state of a microfluidic valve/pump structure. Insuch an embodiment, electrodes could be positioned along the flowchannel on opposite sides of the valve. Application of potentialdifference between the electrodes could reveal not only whether or notthe valve is open or closed, but also the degree of openness of thevalve.

For example, if a potential difference is applied where the valve isshut, no conductive path exists between the electrodes and no currentwill flow if a potential difference is applied. Where the valve ispartially shut, conductive solute would be excluded from a portion ofthe detectable region, altering the current through that region. In suchan embodiment, the detectable entity would in fact be the valve/pumpmembrane.

FIGS. 31 and 32 illustrate particular embodiments of transverse andlongitudinal type electronic detectors in accordance with the presentinvention. However, the present invention is not limited to theseparticular configurations, and other embodiments will be apparent tothose of skill in the art.

For example, where a conductive member is in direct contact with thecontents of the flow (as in the embodiments of FIGS. 31 and 32), chargedspecies in the solute may accumulate at the conductive member. Thisaccumulation of ions can reduce the effective magnitude of theelectrical field applied across the detection region, reducingsensitivity of the detector and complicating measurement of changes inelectrical characteristics.

One way to avoid the effect of accumulation of charged species is toemploy different electrodes for application of the electric field andfor sensing. Accordingly, FIG. 33A shows a plan view of an alternativeembodiment of a “transverse” electronic-based detection apparatus inaccordance with the present invention. FIG. 33B shows a schematicdiagram of the circuit formed by the apparatus of FIG. 33A.

Detector 3300 is similar to that shown in FIG. 31, except that fourelectrodes 3308, 3309, 3310, and 3311 are utilized. An electric field isapplied to detection region 3306 as described above via electrodes 3308and 3310, which function as a source and drain of electrical current.

Additional pair of “sensing” electrodes 3309 and 3311 are used toindependently measure the electric field in the detection region 3306.The magnitude of the sensing voltage is lower than the voltage appliedacross the entire device. Because the voltage sensing electrodes do notsupply or draw a significant current, sensing electrodes 3309 and 3311experience little or no significant ion accumulation effect.

Similarly, FIG. 34 shows a plan view of an alternative embodiment of a“longitudinal” electronic-based detection apparatus in accordance withthe present invention. Detector 3400 is similar to that shown in FIG.32, except that four electrodes 3408, 3409, 3410, and 3411 are utilized.An electric field is applied to detection region 3406 as described abovevia electrodes 3408 and 3410, which function as a source and drain ofelectrical current. An additional pair of “sensing” electrodes 3409 and3411 are used to independently measure the electric field in thedetection region 3406.

While the above description relates to embodiments of detectionstructures and methods utilizing four electrodes, other numbers ofterminals may be employed. For example, three terminal embodimentsoperate in manner similar to the four-terminal embodiments describedabove, except that one terminal serves both to apply the electric fieldand to detect changes in electrical environment. These functions aresegregated between the other two electrodes.

For any of the approaches previously discussed, multiple electrodes canbe positioned along the direction of flow. In such embodiments, thespace between any two electrodes, adjacent or otherwise, defines asensing region. Parallel measurements of the electric field distributionalong the array can increase the sensitivity of detection, and supplyadditional information on the properties of detected entities as afunction of location, elapsed time, and local conditions within themicrofluidic system. Such an embodiment also allows the parallelanalysis of multiple different entities, and or tracking ortime-dependent analysis of given entities.

In transverse embodiments where multiple pairs of electrodes areemployed, at least two electrodes would be positioned on one surface ofthe flow channel at the detection region, and at least two electrodeswould be positioned on an opposing surface at that location. A firstpair of electrodes, consisting of at least one electrode on each ofthese two opposing faces, are used as the source and drain of electricalcurrent, either DC or AC. Two or more additional ‘sensing’ electrodes,consisting of at least a second electrode on each of the opposingsurfaces, are then used to sense the electrical potential across thedetection region. External electrical connections would be arranged suchthese ‘sensing’ electrodes are electrically distinct from the first pairof electrodes.

FIG. 35 shows a simplified plan view of an alternative embodiment of alongitudinal electronic-type detecting apparatus utilizing multipleelectrodes along a flow path. As shown in FIG. 35, detector 3500includes multiple pairs 3502 of electrodes. In such a longitudinalembodiment, one electrode toward the input end, and one electrode towardthe output end are used as the source and drain of electrical current,either DC or AC. At least a second electrode toward either end,electrically distinct from the current source and drain electrodes, arethen used to sense the electrical potential across the detection region.

While the embodiments previously described illustrate detection in aflow channel formed in an elastomer, this is not required by the presentinvention. As described above, embodiments of microfluidic structures inaccordance with the present invention may also include flow channelshaving floors and walls formed in an underlying non-elastomericsubstrate such as glass or silicon, with the ceiling of the flow channelformed from an overlying deflectable elastomer layer. Because thedimensions of the flow channels of nonelastomeric materials in suchalternative embodiments are easily controlled by lithography, thesealternative embodiments are also amenable for use in detecting entities.Moreover, the conductive electrodes could be readily created within orupon the non-elastomeric substrate utilizing techniques such as metalevaporation, sputtering, ion-implantation or chemical vapor deposition.

In addition, while the illustrated embodiments show the electrodespositioned in direct electrical contact with the contents of the flowchannel, this is not required. In alternative embodiments in accordancewith the present invention, the electrodes may be separated from theflow channel by a dielectric material that is of sufficient thickness topermit an AC electrical field to be applied by the electrodes throughthe flow channel.

This encapsulation has at least two advantages. First, the impedancemeasurements become less adversely affected by the presence ofincidental ions in the sample solution. Second, the electrodes canbecome more chemically robust. This enhanced robustness arises fromcreating a physical barrier to electrochemical reaction between theelectrodes and the contents of the flow channel. Encapsulated electrodesmay also be more readily cleanable, in applications where solvents orchemical cleaners are employed.

While FIG. 31 shows the electrodes disposed on opposite sides of theflow channel as in communication with an alternating current (AC) powersupply, the present invention is not limited to this particularconfiguration, and other types of power supplies could be used. In analternative embodiment in accordance with the present invention, a DCpower supply could be connected to the electrodes of FIG. 31. In thiscase, the quantity being measured is resistance rather than capacitance,yet the underlying principle remains the same. An entity is detected andanalyzed through its effect on the impedance of the detection volume. Insuch an embodiment, Coulter-type measurements analogous to the those ofFIG. 32 could comprise the mode of detection.

Many useful characteristics of a detectable entity are frequencydependent. Accordingly, in accordance with yet another alternativeembodiment of the present invention, the frequency of oscillation of theapplied electric field from an AC power supply can be varied duringmeasurement to obtain such frequency dependent information. Possibleexamples of frequency dependent information include permittivity andconductivity of the detectable entity, and electrical characteristics ofthe solute.

While the above embodiments portray the electrodes as disposed on wallsof the flow channel, embodiments in accordance with the presentinvention are not so limited. Other electrode configurations arepossible, for example placement of electrodes in the flow channel floorand ceiling, or even at a flow channel elbow or curve. One significantadvantage of these electronic sensing schemes is that the channel neednot be straight, or of constant dimension, over the entire sensingregion.

2. Magnetic-Based Sensing

Embodiments in accordance with the present invention discussed so farutilize an applied electric field to detect a change in the electricalenvironment of a detection region. In accordance with other embodiments,however, a change in the magnetic environment could be detected toreveal the presence of an entity.

FIG. 36 shows a plan view of one embodiment of such a magnetic-basedsensing device. Sensor 3600 comprises flow channel 3602 fabricated inelastomeric material 3604. A solution including detectable entity 3611flows down flow channel 3602 in the direction indicated.

Sensor 3600 also includes planar spiral conducting member 3608comprising of one or more loops of conductive material, with each endconnected to a lead for external measurement. In one embodiment, planarcoils are incorporated into one or more surfaces of the flow channel.Spiral conducting member 3608 is positioned proximate to flow channel3602, either in walls, floor, or ceiling of flow channel 3602. Spiralconducting member 3608 need not be in direct contact with the contentsof the flow channel.

As detectable entity 3611 flows past spiral conducting member 3608, itsmagnetic properties will induce a voltage or current in spiral 3608.These changes may be monitored to detect the presence of a detectableentity within detection region 3606.

FIG. 37 shows another embodiment of a magnetic-based sensing apparatusin accordance with the present invention. In the embodiment of FIG. 37,detection region 3706 of flow channel 3702 is defined by the presence ofnonplanar coaxial conducting member 3708. Passage of a detectable entityhaving magnetic properties through detection region 3706 may bemonitored in the same manner described above for FIG. 36.

For either of the magnetic-based sensing approaches discussed above,detected entities may be intrinsically magnetic, or magnetizedtemporarily by an external magnetic field applied to the detectionregion. Such an alternative embodiment would permit detection ofspecific magnetic properties of the detectable entity, for exampleparamagnetism, ferromagnetism, and/or diamagnetism.

Yet another embodiment of a magnetic-based sensor in accordance with thepresent invention utilizes magnetoresistive principles. Specifically,FIG. 38 illustrates an embodiment of detector 3800 which incorporates amagnetoresistive sensor 3808 at detection region 3806. The electricalresistance exhibited by magnetoresistive sensor 3808 is sensitive to thepresence of nearby magnetic fields.

Magnetoresistive sensor 3808 is placed adjacent to, or partially within,flow channel 3802 formed in elastomeric material 3804. Magnetoresistivesensor 3808 can, but need not, be, in direct contact with the contentsof the flow channel.

A potential difference from a power supply is applied across sensor3808. The passage of a magnetized detectable entity 3811 throughdetection region 3806 is sensed by measuring the impedance of sensor3808. FIG. 38 shows measurement of impedance by a two-wire method, andFIG. 39 shows measurement of impedance by a four wire measurement.

The various electronic- and magnetic-based sensing described abovediffer in their mechanism for sensing detectable entities, but allfunction by detecting a changed electrical or magnetic environmentwithin a specific detection region. In order to correlate a changedelectrical or magnetic environment in this region with a specific entityor ensemble of entities, a detection volume must be defined.

Specifically, because many of the entities sought to be detected byembodiments of the present invention are of small size, it is importantto define a small enough detection volume such that only one entity, ora particular ensemble of entities, may be present in the detectionvolume and available for detection at a given time. A sufficiently smalldetection volume can be defined in several ways.

One approach is through sample dilution. By making the concentration ofthe sample sufficiently low, the presence of only one detectable entitywithin a given detection volume is ensured.

An alternative approach to defining the detection volume is bycontrolling the physical dimensions of the detection region. By makingthe detection region sufficiently small, the presence of only onedetectable entity is ensured. One way of accomplishing this would be toutilize a constriction in a width of the flow channel.

Embodiments of microfluidic structures in accordance with the presentinvention are particularly suited for defining the detection volumeutilizing this approach. Specifically, the mold defining the width of aflow channel and hence the dimensions of the detection volume can beprecisely controlled at very small dimensions utilizingphotolithographic techniques well known in the art of semiconductorfabrication. Hence, the size and variety of materials that may bedetected within the constriction can be readily controlled duringfabrication of the microfluidic device.

TABLE A listing the range of dimensions of some detectable entities,along with a range of dimensions for the channel at the measurementlocation is given below.

TABLE A APPROXIMATE APPROXIMATE SIZE RANGE OF WIDTH SORTABLE RANGE OFSORTABLE AT DETECTION ENTITY ENTITY (μm) LOCATION (μm) bacterial cell1-10 5-50 mammalian cell  5-100 10-500 egg cell  10-1000  10-1000 spermcell 1-10 10-100 DNA strands 0.003-1    0.001-10   proteins 0.01-1   0.001-10    micelles 0.1-100   1-500 viruses 0.05-1    1-10 larvae600-6500 VARIABLE beads 0.01-100   VARIABLEThe size of the detection region is chosen to optimize the utility ofthe entire device. The dimensions are chosen such that the presence ofthe detected entity causes a readily measurable change in the electricalor magnetic properties of the region, while at the same time permittingthe sample to flow.

As previously described, many electronic-based detection techniques ofthe present invention utilize the application and sensing of an electricfield between electrodes. Accordingly, another approach to defining adetection volume is to adjust the electrode size in order to limit thevolume to which the electric field is applied.

Other approaches to defining detection volume are time based.Specifically, a duration of measurement of the electrical or magneticenvironment may be kept short, so as to limit the number of entitiesentering the detection volume. Alternatively, the rate of flow of sampleinto the detection volume may be controlled, such that the allowableduration for a measurement is within a practical range

While the above description has focused upon detection of a singledetectable entity, the invention is not limited to this approach. Analternative embodiment in accordance with the present invention maydetect ensembles of entities. Such ensembles can include any number ofentities which are either too small, or too numerous in the measurementregion, to be individually detected. Such ensembles of entities would bedetected by their collective effect on the electrical or magneticenvironment of the detection volume. Ensembles of entities for whichthis application could be employed include, but are not limited to,solutions of macromolecules such as nucleic acids and proteins.

3. Electrode Structure and Formulation

Sensing in accordance with electronic- and magnetic-based approaches ofthe present invention may rely upon the use of electrically conductingstructures. Such electrically conducting structures may have anycombination of a number of desirable properties, including but notlimited to mechanical flexibility, adhesion to the surrounding elastomermaterial, resistance to chemical attack, and uniform and low surfaceresistivity.

For example, while the previous Figs. have illustrated conductivestructures in the form of simple, uniform planar electrodes, embodimentsof conductive structures for obtaining data on samples in microfluidicchannels within the scope of the present invention are not limited tothese structures.

Electrically conducting members utilized by embodiments in accordancewith the present invention may assume a wide variety of shapes andsizes. The electrodes may be flexible such that they retain theirelectrically conducting character when physically deformed or stretched.

FIG. 40 shows a plan view of a detection region 4000 in accordance withan alternative embodiment in accordance with the present invention,wherein the conductive structures comprise ‘comb-like’ arrays 4002 and4004. Opposing electrode combs 4002 and 4004 may either face each otheredge-on as shown in FIG. 40, or may feature interlaced combs to form aninterdigitated structure. Such complex electrode shapes enhance thesensitivity of the information collected.

While the previous figures depict simple electrodes having uniformcomposition, this is not required by the present invention. Electrodesuseful with the present invention may have a complex structure featuringregions of high and low conductivity. One example of such a complexelectrode structure would utilize highly conductive regions incombination with regions of intermediate conductivity in order to createregions of homogenous charge.

Such intermediate conductivity regions could be formed utilizing anelastomer incorporating precisely-controlled amounts of conductivematerials such as carbon black, colloidal silver, and charge transfercomplexes such as tetrathiafulavalene/tetracyanoquinodimethane. Suchintermediate-conductivity regions could have a surface resistivity ofbetween about 10⁶-10¹¹Ω.

Moreover, the surface of the electrically conducting members inaccordance with the present invention need not have smooth surfaces.Electrodes or conducting members having textured surfaces can also beemployed to allow flexibility in more than one direction.

The electrically conducting structures utilized by detection apparatusesin accordance with embodiments of the present invention may befabricated in a number of ways. Conducting structures integrated withinan elastomer material in accordance with embodiments of the presentinvention can be fabricated utilizing a variety of techniques. Oneapproach to integrating conducting structures with an elastomericmaterial involves incorporating a conducting polymer within theelastomeric material.

Another approach to electrode formation is to utilize an elastomerbinder that includes electrically conducting materials. In this regard,possible materials include binder materials such as PDMS containingconducting particles such as ZELEC®, manufactured by Milliken Chemicalof Spartanburg, S.C. Examples of other possible candidates ofelectrically conducting materials include VULCAN® carbon black material,manufactured by Cabot Corp. of Alpharetta, Ga. Carbon in the form ofconducting fibrils or nanotubes may also be employed to conveyconductivity for electrode materials in accordance with the presentinvention. For water-based elastomer materials, conductivity can beconferred by the addition of dopants such as iodine or organic saltssuch as potassium iodide,

There are a number of commercially available conductive polymerssuitable for this purpose. These conductive polymers are generallyprovided in a monomer formulation which can be polymerized to conductelectricity.

However, at least one commercial product is sold in polymer form withoutthe need for polymerization. This product is BAYTRON®, manufactured byBayer Corporation of Pittsburgh, Pa. This product can be applied byspraying, spinning, and stenciling techniques. It is also possible topattern BAYTRON® with photolithographic resolution which is suitable formicrofabricated elastomeric channels in accordance with embodiments ofthe present invention.

Still another approach to electrode formation in accordance withembodiments of the present invention is the direct incorporation ofmetals such as gold, silver, or aluminum within the microfluidicstructure. In one embodiment, metal electrode structures may bepatterned upon the elastomer material using chemical or physical vapordeposition techniques, for example. A metallic electrode may be used inconjunction with an intermediate layer to promote adhesion between themetal and elastomer. Alternatively, metals could be physicallyintroduced into the elastomer material, for example by ion implantationor other techniques.

In addition to the use of solid materials as conductive members,alternative embodiments in accordance with the present invention mayalso utilize electrically conducting fluids coated onto elastomermaterials or formed in pockets within the elastomeric structure.Examples of electrically conducting fluids that may serve as electrodesinclude but are not limited to colloidal suspensions of conductingparticles and ionic solutions. The viscosity of the conducting fluid maybe high, for example where conductive greases such as carbon grease orsilver grease are used.

Sensing structures and methods of the present invention are potentiallyapplicable to a broad range of applications. For example, by detectingand characterizing single entities in real time, embodiments inaccordance with the present invention may be employed as sensingelements for sorting devices, an example of which is described inconnection with FIG. 30.

Sensor structures in accordance with the present invention may also beutilized to control flow within a microfluidic device. For example, incertain applications a sample may be flowed against a dialysis membrane.As a result of this flow, concentration of a component of the sample maychange as a function of time or as a function of position along the flowchannel. Where a change in sample component concentration correlateswith a change in the electrical properties to reveal completion of thedialysis process, a valve state may be triggered or pumping may behalted.

Chromatography is another application for detecting changes in sampleimpedance along a flow path. Where the physical position of a sampleafter processing (filtering, separation, dialysis) corresponds to aquantity of interest, the final location of a processed analyte must beidentified. Other potential applications include the sorting by sizeachieved by drawing substances through filters such as bead columns orgels.

Another particular application is for nucleic acid sequencing, where theposition along the stream of analyte may correspond to a particularlength of nucleic acid sequence. Reliance upon distance separationeffects is common to most genomic sequencing efforts, and conventionaldetection of the bands of separated material has been accomplished byoptical interrogation.

Still another application for the instant invention is in monitoringgradient elution that is used to controllably dissociate an analyte fromthe surface of a capillary or column. An example of such a technique isin proteomics, where affinity-based or nonspecific binding to the wallsis systematically blocked through use of a pH or concentration gradientapplied to the contents of a sample channel.

Sensing approaches in accordance with embodiments of the presentinvention method offer an alternative to optical detection of thesize-sorted fragments, by either employing multiple sensing locationsalong the sorted ‘column’ or by recording a time series of data as the‘column’ flows past a particular sensing location.

Sensing methods and apparatuses in accordance with the present inventionare also particularly suited to the analysis of untreated samples, suchas whole blood or environmental fluid samples. The research performed bySohn et al. referenced above includes discrimination of mammalian whiteblood cells from red blood cells, through the electrical properties oftheir DNA content. The ability of a device to discriminate between thesecell types has great potential value.

Microfabricated elastomeric devices in accordance with embodiments ofthe present invention allow fluid handling tasks such as sorting,storage, assaying, and dispensing of analytes to be performed by asingle chip. This integrated character of embodiments in accordance withthe present invention poses an advantage over conventional approachesthat may require one or more washing steps, or direct driving of fluidfrom an external pump. Examples of specific applications for detectionstructures and methods in accordance with the present invention includedetection of pathogens in blood or water samples, antibody bindingstudies in different channel locations, and screening of collections ofcells from cultures or blood samples.

Sensors in accordance with embodiments of the present invention may alsobe employed in conjunction with the “cell cages” described above inFIGS. 26 and 27. In such applications, sensors may be employed tomonitor the position of cells in a cage; to monitor conditions of thesolution surrounding the cells; and to monitor the condition of thecells. The non-intrusive character of detection in accordance withembodiments of the present invention is suited to monitoring changes ina sample that occur over the short term or the long term, from secondsup to weeks or more.

Information that can be obtained utilizing a detector in accordance withthe present invention includes cell size, cell internal resistivity andhence internal cell environment, and cell membrane integrity. Detectionschemes in accordance with embodiments of the present invention may alsoreveal the disposition of the cell within the cage, for example thelocation of the cell and whether or not the cell is bound to the cagesurface. This cellular information can be useful in a variety ofapplications, including but not limited to drug discovery, surfacebioaffinity studies, environmental monitoring, and cell culturing.

Embodiments of detection structures and methods in accordance with thepresent invention also allow for the detection of voids within themicrofluidic channel, such as air bubbles within a fluid sample. Sensingthe presence of such voids may be important in assessing the operationof microfluidic systems. This is especially true for applications whereintake of untreated samples or other field operations can result in theinadvertent inclusion or generation of bubbles within liquid samples.

The application of electronic or magnetic detection methods inaccordance with embodiments of the present invention does not precludeconcurrent utilization of optical sensing techniques. By combiningelectronic and magnetic detection methods sensors described here withestablished optical techniques, new capabilities may be created. Oneexample of such a new capability is to employ sensors in accordance withembodiments of the present invention as non-invasive monitoring devicesused to trigger optical excitation. Such a triggering scheme has thebenefit of reducing the required time of excitation of fluorescent dyesor molecular tags, thereby reducing the probability of photobleachingwithin a particular time period, and prolonging the time beforeoptically-induced sample degradation can be expected to occur.

While the present invention has been described herein with reference toparticular embodiments thereof, a latitude of modification, variouschanges and substitutions are intended in the foregoing disclosure, andit will be appreciated that in some instances some features of theinvention will be employed without a corresponding use of other featureswithout departing from the scope of the invention as set forth.Therefore, many modifications may be made to adapt a particularsituation or material to the teachings of the invention withoutdeparting from the essential scope and spirit of the present invention.It is intended that the invention not be limited to the particularembodiment disclosed as the best mode contemplated for carrying out thisinvention but that the invention will include all embodiments andequivalents falling within the scope of the claims.

What is claimed is:
 1. A method of detecting entities of a selectedentity type in a microfabricated structure, wherein the entity type isselected from bacterial cells, mammalian cells, viral particles,macromolecules, and ensembles of macromolecules, the method comprising:(a) providing a microfabricated structure comprising a plurality of flowchannels each having a detection volume with selected physicaldimensions and width; (b) applying an electric field across thedetection volume; (c) flowing a fluid containing a plurality of entitiesof the selected entity type through the detection volume at a selectedflow rate; (d) measuring a change in current and/or voltage of theapplied electric field resulting from one of the entities in the fluidpassing through and past the detection volume; and (e) correlating thechange(s) with the presence of an entity of the selected entity typewithin the flow channel, wherein the method comprises selecting amicrofabricated structure having the physical dimensions and width ofthe detection volume specified in step (a), in conjunction with a flowrate of the fluid through the detection volume specified in step (c),such that the combination of the physical dimensions and width of theflow channel and the flow rate result in only one entity of the selectedentity type passing through the detection volume at a time.
 2. Themethod of claim 1, wherein the flow channels have a width between 5 and500 microns.
 3. The method of claim 1, wherein the selected entity typeare between 1 and 100 microns in size.
 4. The method of claim 1, whereinthe microfluidic flow channels are formed in an elastomeric material. 5.The method of claim 1, wherein the device is configured to sortmacromolecules detected by the sensing electrodes.
 6. The method ofclaim 1 wherein changes in the current and/or voltage are detected byelectrodes positioned before and after the detection volume.
 7. Themethod of claim 6, wherein the same electrodes are used to apply theelectric field and to detect the change in voltage and/or current acrossthe detection volume.
 8. The method of claim 6, wherein differentelectrodes are used to apply the electric field and to detect the changein voltage and/or current across the detection volume.
 9. The method ofclaim 6, wherein the electrodes are in communication with a DC powersupply such that a constant electric field is applied along thedetection volume.
 10. The method of claim 1, wherein the entity is amacromolecule.
 11. The method of claim 10, wherein the macromolecule isa nucleic acid.
 12. The method of claim 10, wherein the macromolecule isa protein.
 13. The method of claim 1, wherein the entity is an ensembleof macromolecules.
 14. The method of claim 13, wherein the ensemblecomprises nucleic acids.
 15. The method of claim 13, wherein theensemble comprises proteins.
 16. A method of detecting macromolecules ina microfabricated structure, the method comprising: (a) providing amicrofabricated structure comprising flow channels each having adetection volume with predetermined physical dimensions and width; (b)applying an electric field across the detection volume; (c) flowing afluid containing the macromolecules through the detection volume at aselected flow rate; (d) measuring a change in current of the appliedelectric field resulting from a macromolecule passing through and pastthe detection volume; and (e) correlating the change in current with thepresence of a macromolecule within the flow channel, wherein the methodcomprises selecting a microfabricated structure having the width of thedetection volume specified in step (a), in conjunction with a flow rateof the fluid through the detection volume specified in step (c), suchthat the combination of the width of the flow channel and the flow rateresult in only one of the macromolecules passing through the detectionvolume at a time.
 17. The method of claim 16, wherein changes in thecurrent are detected by electrodes positioned before and after thedetection volume.
 18. The method of claim 16, wherein differentelectrodes are used to apply the electric field and to detect the changein current across the detection volume.
 19. The method of claim 16,wherein the macromolecule is a nucleic acid.
 20. The method of claim 16,wherein the macromolecule is a protein.
 21. The method of claim 16,wherein the flow channels have a width between 0.1 and 10 microns. 22.The method of claim 16, wherein the macromolecules are between 0.01 and1 microns in size.
 23. The method of claim 16, wherein the microfluidicflow channels are formed in an elastomeric material.
 24. The method ofclaim 16, wherein the device is configured to sort macromoleculesdetected by the sensing electrodes.
 25. The method of claim 16, whereinchanges in the current are detected by electrodes positioned on oppositesides of the detection volume.
 26. The method of claim 25, wherein thesame electrodes are used to apply the electric field and to detect thechange in current across the detection volume.